Gut bacteria derived microvesicles for vaccine delivery

ABSTRACT

The present invention relates to a vaccine suitable for immunisation against influenza, plague or  Y. pestis  infection said vaccine comprising outer membrane vesicles (OMVs) and the plague vaccine including the V and/or F1 antigens of  Y. pestis.

CROSS REFERENCE TO RELATED APPLICATIONS

This is a U.S. national phase application claiming priority toPCT/GB2019/053007, filed Oct. 22, 2019, which claims priority to Britishapplication 1817194.2, filed Oct. 22, 2018.

The invention to which this application relates is to the delivery ofbiologically active vaccine antigens directly to mucosal sites toinoculate against plague infection and/or influenza using stablemicrovesicles or outer membrane vesicles (OMVs) produced by the humancommensal gut bacterium Bacteroides thetaiotaomicron (Bt).

The production of vesicles derived from the outer membrane of commensalbacteria using recombinant bacteria is disclosed in the applicant'sco-pending application PCT/GB2017/051199 herein incorporated byreference.

Outer membrane vesicles (OMVs) are now known to be naturally producedand secreted by most Gram negative bacteria. Analyses of these 20-400 nmbilayered lipid membrane spherical structures have shown that theycontain major components of the outer membrane such aslipopolysaccharide (LPS) and periplasmic contents of its' ‘parent’bacteria.

Historically, OMVs have been associated with pathogenesis and thestoring and transporting of virulence factors produced by major entericGram negative pathogens including Helicobacter pylori (VacA), Shigelladysenteriae (Shiga toxin) and enterohemorrhagic E. coli (ClyA).

Recently, this paradigm of OMV function has been questioned by evidenceof a non-pathogenic, mutualistic role for OMVs produced by commensal gutbacteria. Members of the genus Bacteroides exclusively packagecarbohydrate and protein hydrolases in OMVs that appear to perform a“social function” in providing substrates for utilization by otherbacteria and contributing to microbiota homeostasis. We have extendedthese observations providing evidence of a broader role of OMVs ingastrointestinal (GI)-tract homeostasis, and the ability ofBacteroides-derived OMVs to influence host immune and epithelial cellresponses.

OMVs contain components that promote their interaction with hostepithelial cells through numerous routes, including micropinocytosis,lipid raft- and clathrin-dependent endocytosis. OMVs produced by B.fragilis contain polysaccharide A that are sensed by dendritic cells viaToll Like Receptor (TLR) 2 leading to enhanced T regulatory cellactivity and production of anti-inflammatory cytokines (IL-10) thatcontributes to protecting the host from experimental colitis [10]. Ourown studies have demonstrated that mammalian intestinal epithelial cell(IEC) intracellular Ca²⁺ signalling is activated by OMVs produced fromthe human commensal bacterium, B. thetaiotaomicron (Bt). We furtherfound that Ca²⁺ signalling was dependent upon a novel constituent of theOMVs: BtMinpp, a homologue of a mammalian inositol phosphatepolyphosphatase cell-signalling enzyme. Collectively, these findingsdemonstrating a non-pathogenic and beneficial role for OMVs produced bycommensal Bacteroides species are consistent with the packaging ofbioactive macromolecules in OMVs to enable members of the intestinalmicrobiota to influence host cell physiology and establish bacteria-hostmutualism.

It is feasible therefore that this pathway of host-microbe interactionmediated by OMVs could be exploited and used as an effective means ofdelivering biologically active proteins to the body and in particular tomucosal sites such as the GI- and respiratory tracts that are vulnerableto injury and disease as a result of exposure to noxious environmentalchemicals and pathogens.

We have undertaken a proof-of-principle study to determine thesuitability of using OMVs produced by Bacteroides thetaiotaomicron (Bt),a prominent member of the intestinal microbiota of all animals, todeliver bacteria-, virus- and human-derived proteins to the respiratoryand GI-tract to protect against infection and tissue inflammation andinjury using mouse models of respiratory influenza infection and wefurther describe the development of a novel drug delivery technologybased upon engineering Bt to express in their OMVs antigens of Y. pestisfor targeted delivery to a non-human primate (NHP) host.

Plague caused by the Gram negative bacterium, Yersinia pestis, is anancient disease, accounting for many deaths over hundreds of years andstill exists in parts of the world today. To protect against infectionvaccines need to be able to elicit both humoural immunity andneutralising antibodies and cell-mediated immunity that is effective atprimary, mucosal, sites of infection [1, 2].

There is currently no licensed plague vaccine in the Western world.Previously available US Pharmacopeia killed whole cell vaccines providedprotection against bubonic but not pneumonic plague but due tounacceptable reactogenicity were discontinued [3]. Live-attenuatedvaccines have been used in countries of the former Soviet Union andChina although due to unacceptable reactogenicity to the vaccine andrisk of reversion to full virulence they have not been licensed for useelsewhere including the USA [4]. The Fraction 1 (F1) and LcrV(virulence; V) Y. pestis proteins encoded by the Fra/pMT1 and pYVplasmids respectively [5], have been identified as major protectiveantigens that are essential for preventing phagocytosis of the bacteriaand regulating type three secretion, respectively [6]. The presentemphasis on developing F1 and V based vaccines is on recombinantprotein-based subunit vaccines (rF1V) that incorporate chemicaladjuvants. These can provide good protection in pre-clinical animalmodels [7, 8] although the F1-V fusion vaccine does not protect againstF1 strains with modifications to the type III secretion system, and theduration of protection against pneumonic infection is also uncertain[9]. Injections and the use of needles for delivering these and othercurrent vaccines has the associated risks of contamination, lack ofpatient compliance and high cost of mass immunisation, and a requirementfor cold chain delivery and storage. Importantly, injected vaccines mayalso provide partial or no protection at primary, mucosal, sites ofplague infection [2, 10]. Collectively these issues constrain the use ofexisting plague vaccines particularly in resource-poor low incomesettings.

Another approach to developing more effective mucosal vaccines forpathogens such as plague is using nanoparticle-based platforms. Theseinclude virus like particles, immune stimulating complexes, polymericnanoparticles, inorganic nanoparticles, liposomes, and emulsions thathave the capability of overcoming the high production costs and safetyconcerns of live vaccines in addition to the often weak immunogenicityand adjuvanticity of subunit and recombinant protein based vaccines[10]. These nanoscale carrier technologies enable conformationallycorrect antigens to be incorporated into highly stable nanoparticlesthat can control the spatial and temporal presentation of antigens tothe immune system leading to their targeted and sustained release. Anoverlooked component of platform nanoscale vaccines are bacterialmicrovesicles and in particular, outer membrane vesicles (OMVs) ofGram-negative bacteria [11]. While many synthetic nanoparticles arecapable of transferring heterologous antigens to antigen presentingcells (APC), the ability to efficiently stimulate the immune system isoften not inherent [12]. OMVs, however, can combine high stability withantigen presentation and native adjuvanticity, making them an attractivevaccine platform [13].

Vesiculation and outer membrane vesicle (OMV) production is afundamental characteristic of Gram-negative bacteria unrelated tobacterial lysis or membrane instability that full fills key requirementsof a prokaryotic secretion process [14]. Nanoscale OMV proteoliposomescontaining immunogenic components derived from the bacterial outermembrane and periplasm target APC, including dendritic cells [15-17]leading to T cell and B cell immunity. Research with OMVs frompathogenic bacteria including Neisseria meningitides and Vibrio cholerasupports the case of OMVs as vaccine candidates [18] with those derivedfrom N. meningitides having proven safety and efficacy records incontrolling serogroup B meningococcal (MenB) disease outbreaks [19, 20].Thus, OMV based vaccines offer significant advantages over conventionalvaccines; they are non-replicating, provide needle-free delivery, targetmucosal sites, have an established safety record, can elicit innate andantigen-specific adaptive immune responses, and possess self-adjuvantproperties (i.e. microbe associated molecular pattern molecules [MAMPs]such as lipopolysaccharide [LPS]). The limitations of current,pathogen-derived, OMV vaccines are the potential for unintended toxicitydue to associated toxins, low expression levels of protective antigens,variable efficacy depending on source and formulation, the need forexogenous adjuvants, and providing only incomplete protection because ofstrain variation. These limitations could in principle be overcomethrough the use of non-pathogenic OMV-producing commensal bacteria,engineered to improve their vaccine application. The recentdemonstration that OMVs produced by the prominent human commensal gutbacteria Bacteroides thetaiotaomicron (Bt) access and influenceintestinal epithelial and immune cells [21, 22] supports thisproposition and identifies a means by which commensal gut bacteria caninfluence host cell physiology.

It is therefore an aim of the present invention to provide a vaccine orinoculation effective against plague or Y. pestis infection whichaddresses the abovementioned problems.

It is a second aim of the present invention to provide a method ofproducing a vaccine or inoculation which addresses the abovementionedproblems.

It is a third aim of the present invention to provide a method ofvaccination or inoculation against plague or Y. pestis infection.

It is therefore a further aim of the present invention to provide avaccine or inoculation effective against influenza infection.

It is a yet further aim of the present invention to provide a method ofproducing an influenza vaccine or inoculation which addresses theabovementioned problems.

It is a yet further aim of the present invention to provide a method ofvaccination or inoculation against influenza infection.

In a first aspect of the invention there is provided a vaccine suitablefor immunisation against plague or Y. pestis infection said vaccinecomprising outer membrane vesicles (OMVs) including the V and/or F1antigens of Y. pestis.

Typically the OMVs are produced by gram-negative bacteria. Furthertypically the gram-negative bacteria are human commensal gut bacteria.

Preferably the OMVs are produced by bacteria from the genus Bacteroides.Further preferably the OMVs are produced by Bacteroides thetaiotaomicron(Bt).

Typically genes or mini-genes encoding the V and/or F1 Y. pestisproteins were cloned downstream of sequences encoding the N-terminalsignal peptides of the OMV protein OmpA (BT_3852). Further typically theprotein products are contained within the lumen or outer membrane ofOMVs.

Typically the gene constructs are generated in E. coli hosts and thenintroduced into Bt.

In one embodiment a synthetic gene construct of 1043 bp encoding the Vantigen and/or a synthetic operon construct of 3826 bp encoding caf1M,caf1A and/or caf1 genes of the caf1 operon that generates the F1 proteinwere N-terminally fused to the OmpA signal peptide of Bt. Typically thiscreates a construct in silico with codon usage being optimised forexpression in the same species.

In one embodiment signal peptide prediction is used.

In one embodiment the resulting gene cassettes were obtained throughgene synthesis and subsequently cloned into the E. coli plasmid pEX-A2and pEX-K4 respectively.

Typically the cassettes contain BspHI and/or EcoRI restriction sites attheir 5′ and 3′ ends, respectively. Typically the genes encoding V1and/or F1 were excised from the pEX derivatives using BspHI and/orEcoRI. Further typically the genes were ligated into theNcoI/EcoRI-restricted pGH090 expression vector, resulting in pGH179 andpGH180 respectively.

In one embodiment the V and/or F1 containing OMVs have an average sizeof substantially 400 nm.

Typically, the V and/or F1 containing OMVs exhibit thermostability.Further typically the OMVs exhibit minimal loss of vaccine antigencontent after storage for 6 weeks at 4° C. or 40° C.

In one embodiment the vaccine is delivered to the gastrointestinal (GI)tract. In a preferred embodiment the vaccine is delivered to therespiratory tract.

In a second aspect of the invention there is provided a method ofproducing a vaccine or inoculation, said method including the step ofintroducing at least part of the gene sequence encoding V and/or F1antigens of Y. pestis into a gene sequence for OMV production.

In a third aspect of the invention there is provided a method ofvaccination or inoculation against plague or Y. pestis infection, saidmethod including the steps of introducing an OMV including the V and/orF1 antigens of Y. pestis to the body.

Typically oral and/or nasal administration are the preferred routes ofvaccination. Further typically this generates protective immunity atprimary sites of plague infection.

In one embodiment for oral delivery a dose of 50 μg of V antigen wasused. Preferably the dose was formulated in Bt OMVs.

In a further aspect of the invention there is provided a vaccinesuitable for immunisation against influenza infection said vaccinecomprising outer membrane vesicles (OMVs) characterised in that withinand/or on the outer membrane of Bt OMVs both bacteria and/or virusderived vaccine antigens are delivered in a form capable of elicitingantigen specific immune and antibody responses in mucosal tissues and/orsystemically.

In one embodiment BT OMV is produced using a synthetic gene construct.Typically the gene construct encodes a synthetic influenza strain. Inone embodiment A 635 bp synthetic gene construct encoding a syntheticinfluenza (H5F; from IAV strain H5N1 (VN/04:A/VietNam/1203/04)) is used.

Typically, pre-fusion headless HA mini-stem N-terminal is fused to theOmpA signal peptide of Bt is created in silico.

Typically the resulting gene cassette is obtained through gene synthesisand subsequently cloned into E. coli. Further typically the E. coliplasmid pEX-K168 (Eurofins, Germany) is used.

In one embodiment the cassette contains BspHI and EcoRI restrictionsites at its 5′ and 3′ ends, respectively. This allows allowing for thetranslational fusion of the gene to the start codon in the Bacteroidesexpression vector pGH090. Typically the gene is excised from pEX-K168using BspHI and EcoRI and ligated into the NcoI/EcoRI-restricted pGH090expression vector, resulting in pGH184.

Specific embodiments of the invention are now described with referenceto the following figures:

FIG. 1. Bt OMV plague vaccines. A. Schematic of cloning procedure forthe expression of Yersinia virulence proteins F1 and V at the surface orin the lumen, respectively, of Bt OMVs. The Bt secretion signal sequenceis indicated in yellow and is fused at the N-terminus of the F1 and Vgenes. B. Expression of V antigen in OMV lysates determined byimmunoblotting (IB). C. Determination of protein location aftertreatment with proteinase K (PK). IB of V antigen with and withoutpre-treatment of V-OMVs or recombinant V (rV) with proteinase K. NT: nottreated. D. Thermostability of V-OMVs assessed by storing preparationsat different temperatures for 8 weeks prior to analysing OMV extracts(E) or storage buffer (B) for presence of V antigen by IB. Molecularweight (MW) is expressed in kDa. E. Size distribution of Bt V-OMVs bynanoparticle tracking analysis. The OMV suspension was diluted 100times. F. Schematic of OMV-plague vaccine NHP immunisations via theintranasal (IN) or oral route and analyses.

FIG. 2. Systemic and humoral response to F1-OMV vaccines. Serum (A) andsaliva (B) from animals prior to (pre) and 56 days post immunizationwith F1-OMVs were analysed for F1-specific IgG and IgA antibodies,respectively by ELISA. Bronchoalveolar lavage fluid (BAL) (C) andsalivary gland homogenates (D) were analysed for antigen-specific IgA atthe study end point. The data shown represents mean±SEM values.

FIG. 3. Humoral systemic immune response to V-OMV vaccines. The amountsand titre of V-antigen specific IgG in the sera of animals immunisedwith V-OMVs via the intranasal or oral route was determined prior toimmunisation (day −14 and day 0) and at three different timepoints postimmunisation (days, 28, 42 and 56) by ELISA using an initial serumdilution of 1:10 prior to serial 1 in 4 dilutions. Each line graphrepresents the mean value for each group (n=4). The levels of V-specificIgG in individual animals immunised with 25 mg of V-OMVs via theintranasal route is shown in the bottom right graph with the horizontallines in each category representing the mean±SEM.

FIG. 4. Mucosal humoral immune response to V-OMV vaccine. A. The amountsand titre of V-antigen specific IgA in the saliva of animals immunisedwith V-OMVs via the intranasal or oral route was determined prior toimmunisation (day 0) and at three different timepoints post immunisation(days, 28, 42 and 56) by ELISA using serial 1 in 2 dilutions. Each linegraph represents the mean value for each group (n=4). B. Levels ofV-specific IgA in saliva samples of individual animals immunised with 25μg of V-OMVs via the intranasal route. The horizontal lines in eachcategory representing the mean±SEM. C. Levels of V-specific IgA insalivary glands and bronchoalveolar lavage (BAL) samples for animalsimmunised intranasally or orally with V-OMVs analysed at day 56 postimmunisation.

FIG. 5. Cytokine production by PBMCs from V-OMV immunized animals. PBMCsobtained from animals previously immunised with V-OMVs via theintranasal (IN) or oral at day 56 post immunisation were cultured in thepresence or absence (PBS) of recombinant V protein (rV) for 72 hoursafter which supernatants were analysed for cytokine content using amultiplex bead assay and flow cytometry. Limit of detection for givenvalues was IL-1β <1.11 pg/mL, MCP-1 <1.61 pg/mL, IL-6 <1.45 pg/mL, IL-8<1.02 pg/mL, IL-23 <1.44 pg/ml. Given values represent the groupmeans±SEM of duplicate samples, n=4 per group. *p≤0.05, ***p≤0.001,****p≤0.0001.

FIG. 6. Principal coordinates analysis for weighted UniFrac distancemetrics for 16S rRNA sequences obtained from animals immunised withV-OMVs to visualise the degree of relatedness between samples. A. Theblue symbols represent the faecal microbiotas (blue) of individualanimals immunised with V-OMVs via the oral route and the red symbolsrepresenting the nasal microbiota of animals immunised with V-OMVs viathe intranasal route. B. The nasal microbiotas of animals pre-(circles)and post (squares) V-OMV intranasal immunisation. The colours indicatedifferent doses of V-OMVs.

FIG. 7. A competitive ELISA assay to assess the functionality of immunesera from V-OMV immunised animals. Serial dilutions of sera from animalsimmunised with V-OMVs identified by individual code numbers shown in thekey were tested for their ability to displace a monoclonal V antibodyfrom the surface of the V antigen immobilised in microtitre plate wells.Reference serum was from NHPs immunised intramuscularly with recombinantF1 and V plus adjuvant.

FIG. 101. Size, structure and stability of Bt OMVs. (a) Electronmicroscopy (EM) of Bt cells showing vesicles budding from their surfacebefore release into the milieu (lines in left panel), and EM image ofOMVs extracted from cell culture supernatant (right panel). (b)Immunodetection of native Bt OMVs using colloidal gold anti-rabbit Ig todetect binding of rabbit anti-Bt OmpA antisera (right panel). Left panelshows absence of staining of OMVs produced by an OmpA deletion mutant ofBt. (c) Size distribution of OMVs produced by Bt determined bynanoparticle tracking analysis. (d) The thermostability of OMVs wasassessed after storage of OMV suspensions at 4° C. or 40° C. for 30days. Immunoblotting for the detection of OmpA was performed on OMVextracts and storage buffer (SB).

FIG. 102. Expression of heterologous proteins in Bt OMVs. (a) Schematicof cloning procedure for the secretion of proteins of interest in thelumen or at the surface membrane of OMVs. The secretion peptide of BtOmpA (SP BtompA) is indicated in yellow and fused at the N-terminus ofthe gene of interest. (b) Determination of protein location aftertreatment with proteinase K (PK). Immunoblotting of STOmpA and KGF-2with and without pre-treatment of OMV suspensions with proteinase K. NT:not treated; PK: + Proteinase K; B: PK buffer alone. (c) KGF-2quantification within OMVs. Comparison of KGF-2 pure protein (1-100 ng)with 10 μl of 1 ml OMV suspension (S).

FIG. 103. Intrinsic adjuvanticity of Bt OMVs. Mice (n=5) wereintranasally administered native PBS alone (a) or Bt OMVs in PBS (b) and5 days later upper and lower respiratory tract tissue was processed forimmunohistology to visualise immune cell activation and formation oforganised lymphoid tissue containing CD45R⁺ B cells, CD3⁺ T cells andIba-1⁺ dendritic cells (DC) in the nasal associated lymphoid tissue (a,b and d), the inducible bronchus-associated lymphoid tissue (iBALT) (c),and fat associated lymphoid clusters (FALC) within the lung (d).

FIG. 104. Bt OMV-elicited systemic and mucosal antibody responses. (a)Mice (n=5-6/grp) were administered Bt OMVs expressing the SalmonellaOmpA or SseB proteins via the oral (OG), intranasal (IN) orintraperitoneal (IP) routes according to the dosing regimen described inthe Material and Methods section. At autopsy, serum (b) andbronchoalveolar lavage fluid (BAL) (c) were analysed for anti-OmpA andanti-SseB IgG and IgA antibody titres, respectively, by ELISA. Theboxplots identify the mean and upper and lower quartile values for datasets obtained from animals within each treatment group. Analysis ofvariance for multiple comparisons of means between independent samples(ANOVA) was followed by a Tukey's test. ***P<0.001; ns=non-significantdifferences. Total IgA levels in BAL (d) and salivary gland (e) samplesfrom animals treated with StOmpA OMVs or PBS (control) were determinedby ELISA using IgA standards as described in the Materials and Methodssection.

FIG. 105. Bt OMVs expressing IAV H5F protein confer a level ofprotection to virus infection. Mice were immunised intranasally withH5F-OMVs in PBS, or as controls wild type OMVs or PBS alone and 28 dayschallenged intranasally with a lethal dose of PR8 virus. The weight ofindividual animals was assessed daily and at necropsy serum andbronchoalveolar fluid (BAL) were analysed for IAV or H5 specificantibodies by ELISA. Lung homogenates were assessed for viral load(PFU). NS, no significant; *P<0.05; **P<0.01; ***P<0.001.

FIG. 106. OMVs containing KGF-2 ameliorate DSS-induced colitis. (a)Groups of mic were provided drinking water with or without2.5% (w/v) DSSfor 7 days. On days 1, 3 and 5 mice were orally gavaged with either PBSalone, native OMVs or OMVs containing KGF-2. (b) Percent weight loss atday 7. (c) Colon length at day 7. (d) Disease Activity Index (DAI) atday 7. (e) Representative images of colons. Data expressed as mean±SD(n=5). Mice gavaged with PBS receiving regular drinking water wereconsidered as the reference group for statistical analysis. NS, nosignificant; *P<0.05; **P<0.01; ***P<0.001.

FIG. 107. OMVs containing KGF-2 protect and restore goblet cells inDSS-induced colitis. (a) Histological score of colon tissue asdetermined by microscopy of H&E stained sections. (b) Number of AlcianBlue stained goblet cells per mm² of epithelial area. (c) Microscopeimages of goblet cell distribution in representative colon sectionsstained with Alcian Blue. Data expressed as mean±SD (n=5). *P<0.05.

Figure S1. Acquisition of fluorescent labelled Bt OMVs by lungmacrophages and their subsequent trafficking to cervical and mediastinallymph nodes after intranasal administration.

Figure S2. The impact of orally administered Bt OMVs on the recipientsintestinal microbiota. Mice (n=5) were oral gavaged with native OMVs onday 3, 5 and 7, and faeces collected on day 0, 4, 7 and 8 to evaluatethe impact of the OMVs on the host microbiota. Faeces were weighed,homogenized in PBS and serially diluted prior to plating on differentagar media: Nutrient=total aerobes, Wilkins-chalgren=total anaerobes,Brain Heart Infusion (BHI)=total anaerobes and BHI supplemented withgentamicin and amikacin=Bacteroides. The results are expressed in thelogarithm of the CFU normalized to the weight of individual faecalsamples for each day and growth medium. Data is expressed as mean±SD.Statistical significance differences were evaluated using Dunnettbilateral post-hoc to compare days after OMV administration vs. thecontrol day 0. *P<0.05; **P<0.01.

Figure S3. Colonization of OMV-StOmpA immunised mice after Salmonellachallenge. Mice immunised with Bt StOmpA-OMVs via the oral or parental(intraperitoneal; ip) route or that were orally administered native OMVs(see Materials and Methods for immunisation protocol) were challengedwith an oral dose of 10⁸ CFU Salmonella typhimurium SL1344 and 5 dayslater the animals were euthanised and the bacterial load in the ileumand colonic contents, and homogenates of mesenteric lymph nodes (MLN),ileum tissue, spleen and liver were determined by plating serialdilutions on xylose lysine to deoxycholate agar plates supplemented with50 μg/ml streptomycin. The data shown represents sample CFU values forindividual animals.

Figure S4. Evaluating the biological activity of KGF-2 contained in BtOMVs using an epithelial wound-healing assay. (a) Representativemicrographs of healing of a scratch wound in a confluent monolayer ofCaco2 cells after exposure to PBS, naïve OMVs, KGF-2 OMVs or recombinantKGF-2 for 72 h. Red dotted lines demarcate the wound margin. (b)Graphical representation of cell growth across the wound area after 72 has determined by pixel².

Referring firstly to the plague vaccine, plague caused by the Gramnegative bacterium, Yersinia pestis, is still endemic in parts of theworld today. Protection against pneumonic plague is the paramountrequirement to prevent epidemic spread yet there is currently nolicensed plague vaccine in the Western world. Here we describe the meansof delivering biologically active plague vaccine antigens directly tomucosal sites of plague infection using highly stable microvesicles(outer membrane vesicles; OMVs) naturally produced by the prominent andharmless human commensal gut bacterium Bacteroides thetaiotaomicron(Bt). Bt was engineered to express in their OMVs the major plagueprotective antigens Fraction 1 (F1) in the outer membrane and LcrV (V)in the lumen for targeted delivery to the gastrointestinal (GI) andrespiratory tracts in a non-human primate (NHP) host. The key findingsof our study are that Bt OMVs stably expresses F1 and V plague antigens,and in particular the V antigen, in the correct, immunogenic form.V-OMVs delivered intranasally elicit substantive and specific immune andantibody responses both in the serum (IgG) and in the upper and lowerrespiratory tract (IgA), including the generation of serum antibodiesable to kill plague bacteria. Our results also show that Bt OMV basedvaccines posses many desirable characteristics of a plague vaccineincluding biosafety and absence of any adverse effects, pathology orgross alteration of resident microbial communities (microbiotas), highstability and thermo-tolerance, needle-free delivery, intrinsicadjuvanticity, the ability to stimulate both humoral and cell mediatedimmune responses, and targeting of primary sites of plague infection.

Nasal administration are the preferred routes of vaccination to generateprotective immunity at primary sites of plague infection. To beeffective, significant challenges including minimising loss ofimmunogenicity during transit and optimising dosing regimens and routesof delivery that safely generates protective antibodies must be met. Toovercome these challenges we have developed a novel drug deliverytechnology platform that exploits the natural production of nanoscalemicrovesicles called outer membrane vesicles (OMVs) by the prominenthuman commensal gut bacterium Bacteroides thetaiotaomicron (Bt). Using anon-human primate model we have shown that Bt OMVs expressing the majorplague protective antigens F1 and V when delivered nasally elicitabundant and specific antibodies in both the serum (IgG) and upper andlower respiratory tract (IgA) including antigen-specific IgG antibodiesthat were active in two independent surrogate assays of protection. Ourresults also highlight key advantages our Bt OMV vaccine technologyoffers over current plague vaccines in terms of technology and/orapproach. These include their acellular non-infectious nature, needlefree delivery, direct targeting of primary sites of mucosal plagueinfection, intrinsic adjuvanticity, activation of both the innate andadaptive arms of the immune system, and have no cold chain requirement.

Here we describe development of a novel drug delivery technology basedupon engineering Bt to express in their OMVs the V and F1 antigens of Y.pestis for targeted delivery to the gastrointestinal (GI) andrespiratory tracts in a non-human primate (NHP) host. Our findingsdemonstrate OMV-plague vaccines are an effective means of eliciting bothmucosal and systemic antibody responses and systemic cell-mediatedresponses with delivery of OMV vaccines by the respiratory route beingparticularly effective at eliciting antigen-specific IgG antibodies thatwere active in two independent surrogate assays of protection.

Results

Study Design and Rationale

The aim of this feasibility study was to determine the suitability ofOMVs produced by the human commensal gut bacteria, Bt, as a plaguevaccine antigen delivery platform to activate mucosal and systemicimmune responses in a non-human primate (NHP) host with the capabilityof protecting against plague infection. Although mice are a commonexperimental model system used in preclinical studies of human drugs andvaccines, the NHP is better suited to assess the particular questionsconcerning Bt OMV vaccine efficacy and safety as their respiratory andGI-tracts and natural diet (and hence microbiome) are far closer to thatof humans. Their use therefore de-risks the development pathway forBt-OMV vaccine by providing assurance that the NHP microbiome andhistological integrity of the GI-tract and other associated tissues arenot adversely affected after immunisation. In addition, the use of NHPantisera to demonstrate bactericidal activity would help pave the wayfor assessment of the protective effect of Bt OMV vaccination in humansas a surrogate for protection against infection.

Expression of Y. pestis Vaccine Antigens in Bt OMVs

Mini-genes encoding the V and F1 Y. pestis proteins were cloneddownstream of sequences encoding the N-terminal signal peptides of themajor OMV protein OmpA (BT_3852) whose products are contained within thelumen or outer membrane of OMVs (FIG. 1A). The constructs were generatedin E. coli hosts and then mobilised into Bt via a triple filter matingprotocol using a helper strain. Immunoblotting of whole cell and OMVlysates of recombinant Bt strains confirmed expression of the V antigen(FIG. 1B). Whilst it was not possible to detect the F1 protein byimmunoblotting, we were able to confirm its expression by LC-MSproteomics of OMV lysates (data not shown). The luminal versus outermembrane distribution of the proteins in Bt OMVs was established using aprotease protection assay which showed that V protein distribution waswithin the lumen of OMVs (FIG. 1C) whereas F1 expression was associatedwith the OMV outer membrane (data not shown). V and F1 containing OMVshad an average size of ˜400 nm (FIG. 1E) and exhibited highthermostability with minimal loss of vaccine antigen content afterstorage for 6 weeks at 4° C. or 40° C. (FIG. 1D and data not shown).

OMV Immunisation Protocol

Oral and nasal administration are the preferred routes of vaccination togenerate protective immunity at primary sites of plague infection [9].We initially set out therefore to determine which of these routes wasoptimal for Bt OMV-plague vaccines as determined by measuring both localand systemic antigen-specific V and F1 IgA and IgG antibodies. For oraldelivery we used a dose of 50 μg of V antigen formulated in Bt OMVs,which is mid-range of vaccine dose used previously in cynomolgusmacaques [26] and is a human-equivalent dose and concentration [7]. Inconsidering the potential risks of administering agents via theintranasal route and its accessibility to the systemic circulation andthe brain, we used a range of OMV vaccines doses (12.5, 25 and 50 μg) toassess safety, tolerability and determine the lowest dose required toinduce a strong immune response. OMV vaccination followed a prime andsingle boost dosing regimen as depicted in FIG. 1F.

Host Response to F1-OMV Plague Vaccines

F1-OMV vaccine formulations were evaluated by measuring antigen-specificIgA and IgG levels in mucosal secretions and tissues and in the serum,respectively (FIG. 2). F1-OMVs generated antigen-specific IgG serumantibodies (˜0.5-1.5 μg/ml) after both oral and intranasal immunisationwith evidence of inter-individual variation in levels of F1 specific IgGin most experimental groups; whereas one animal administered 12.5 μgF1-OMVs intranasally failed to respond another had high levels ofreactive antibodies prior to immunisation, at day −14, although thisreactivity was no longer evident at day 0, returning to levelscomparable with other animals. There was no evidence of an antigen doseor dependency in the levels of antibodies produced as similar levels ofF1 specific IgG were seen in animals receiving 12.5, 25 or 50 μg ofF1-OMVs (FIG. 2a ). There was also no clear evidence for the superiorityof oral versus nasal delivery of F1-OMVs in terms of levels of antigenspecific IgG antibodies generated (FIG. 2A). F1-OMVs elicited weakmucosal immune responses with low levels of antigen specific IgA presentin saliva (FIG. 2B) and BAL (FIG. 2C) samples irrespective of the routeof administration. It was not possible to detect F1-specific IgAantibodies in the salivary glands of F1-OMV immunised animals (FIG. 2D).

Host Response to V-OMV Plague Vaccines

V-OMV vaccines generated strong antibody responses systemically and atmucosal sites (FIG. 3). Analysis of serum anti-V specific IgG antibodiesshowed that the intranasal route of V-OMV immunisation generated highertitres of antibodies compared to oral immunisation (FIG. 3); contrastingwith the findings from F1-OMVs immunisations demonstrating no vaccinedelivery route related differences in IgG responses (FIG. 2). Thehighest titres of V-specific IgG at the study end point were in animalsintranasally vaccinated with 25 μg of V-OMV with antibody levelsincreasing over the study period (FIG. 3). In mucosal samples, lowtitres of V-specific IgA were detected in the saliva at all time pointsanalysed (FIG. 4A) with the highest titres seen at day 42 (FIG. 4B).Consistent with the superior performance of intranasally deliveredV-OMVs for generating V-specific IgG antibodies, higher levels ofV-specific IgA were seen in the saliva of animals immunised intranasallycompared to those immunised via the oral route (FIG. 4A, C). The 25 μgdose of intranasally administered V-OMVs produced the highest titres ofV-specific IgA in saliva (FIG. 4A), similar to serum V-specific IgGantibody responses (FIG. 3). By comparison, salivary gland V-specificIgA antibody titres at day 56 were equivalent in animals immunised with12.5, 25 or 50 μg of antigen (FIG. 4C). The titre of V-specific IgAantibodies in the BAL were lower than that in both the saliva andsalivary glands with no evidence of vaccine dose-level dependentresponses as each dose of V-OMVs elicited similarly low levels ofV-specific IgA (FIG. 4C).

As an indicator of cell mediated immune responses to OMV vaccines weanalysed the recall response of peripheral blood lymphocytes fromanimals immunised intranasally or orally with V-OMVs afterre-stimulation with rV antigen in vitro. PBMCs from V-OMV immunisedanimals constitutively produced varying levels MCP-1, IL-6, IL-8 and/orIL-23 during culture in complete media alone (FIG. 5). In the presenceof rV the levels of these cytokines were upregulated in all PBMC samplesirrespective of the route or dose of V-OMVs used for immunisation. Inaddition, IL-1□ production which was absent in control, non-stimulatedcultures, was induced by V antigen re-stimulation of PBMCs (FIG. 4).Other cytokines included in the analysis that were not detected in anyPBMC sample or were present at levels below the detection limit of theassay (≤1.0 pg/ml) included IFN□, TNF□, IL-10, IL-12p70, IL-17A, andIL-18 (data not shown).

Safety of V-OMV Vaccines

The biosafety evaluations of F-OMV and V-OMV vaccines were based onhistopathology of tissue recovered at necropsy, and profiling residentmicrobe populations (microbiotas) of the GI- and respiratory tractsusing 16S rRNA sequence-based community profiling of faecal- and nasalswab-derived DNA samples, respectively, pre- and post OMV immunisation(FIG. 6). Independent, blinded evaluation of various tissues (lung,spleen, liver, heart, lymph nodes, kidney, brain and regions of theGI-tract) at necropsy revealed no macroscopic signs of pathogenicinfection or pathology.

Evidence of recent immune activation in the lymphoid tissues of thespleen and lymph nodes in a proportion of animals in each group ofF1-OMV animals was seen; this comprised the presence of scatteredsecondary follicles with mitotic figures and apoptotic cells in thesplenic white pulp and cortex of the lymph nodes. Microscopicexamination of tissues from animals receiving the highest dose of V-OMVs(50 μg) identified regions of organised and enlarged lymphoid structuresand follicles within the spleen and lungs in the absence of anyinfection or bacteria (data nor shown). Lympho-plasmacytic cellinfiltrates were observed with some frequency in the mucosa of all partsof the GI tract; this is a common finding in non-human primates, andtheir presence likely reflects a low-grade, chronic-activegastritis/enteritis/colitis which may or may not be associated withclinical signs such as diarrhoea. Filamentous bacteria have beenobserved previously in the stomach of captive-bred macaques andBalantidium infection is a common, incidental finding in the caecum andcolon of non-human primates, and is usually asymptomatic.

16S rRNA community profiling and PCA analysis of sequence data revealedlittle inter-individual differences in the faecal microbiota at baselineand prior to OMV vaccine immunisation (FIG. 6A). By contrast, there wasconsiderable inter-individual variation in the nasal microbiotas ofdifferent animals at baseline (FIG. 6A). With the exception of one ortwo samples intranasal immunisation with V-OMVs did not noticeably alterthe profiles at any vaccine dose (FIG. 6B) with the sequence data setsfrom pre- and post-immunised animals clustered closely together. Thesefindings indicate that V-OMVs have no major impact on resident microbe(prokaryote) communities in the upper respiratory tract.

Functionality of OMV-Elicited IgG Antibodies

Two independent assays were used to assess the functionality of immunesera from OMV immunised animals and to determine their usefulness aspotential immune correlates of protection in humans. The first assayused was a competitive (CE)-ELISA [1] that quantifies the ability ofimmune IgG to compete for binding to the Y. pestis V antigen with amonoclonal antibody (Mab 7.3) which can protect mice by passive transferagainst plague infection [27]. The second assay is a novel bactericidalassay specifically developed for this study that assesses the level ofantibody and complement killing of Y. pestis in serum samples using theY. pestis reference strain CO92 as the target.

Serum samples collected at the study endpoint from representativeanimals within each of the different route of administration anddose-level groups were assayed for their ability to displace Mab 7.3from binding to rV in vitro. The data are presented as a titration linefor loss of binding of the mouse monoclonal antibody with increasedconcentration of test samples (see Materials and Methods) using as areference, macaque immune sera obtained by parenteral immunization withrF1+ rV proteins (FIG. 7). The sera from animals intranasallyadministered V-OMVs at all doses inhibited to some extent the binding ofMab 7.3, which at the higher serum concentrations were comparable to theactivity of the reference sera. Sera from animals immunized with 25 or50 μg of V-OMVs contained the highest titre of competitive antibodies(FIG. 7). By comparison, sera from animals orally administered V-OMVscontained low or no antibodies capable of competing with Mab 7.3 forbinding to V antigen.

The bactericidal assay provided a functional activity assessment of the48 separate test groups of samples taken from immunised cynomolgusmacaques. Throughout the assays conducted, the reference antisera(generated by immunising macaques with recombinant rF1 and rV) providedconsistent dose-response bactericidal activity (BCA) behaviour (Table1).

TABLE 1 Summary of serum antibody bactericidal assay outputs (ED50 inunits of % serum^(†)) Dose Day Day Day Day Vaccine (μg) Route 0 28 42 56OMV-F1 50 IN 12.1 36.2 14.9 >45 OMV-V 50 IN 0.6 5.5 0.8 14.9 OMV-F1 25IN 1.4 1.6 1.5 6.2 OMV-V 25 IN 1.9 0.5 15.3 13.7 OMV-F1 12.5 IN >4521.6 >45 39.2 OMV-V 12.5 IN >45 >45 20.9 22.5 OMV-F1 50 OG >45 17.013.0 >45 OMV-V 50 OG >45 1.8 6.3 >45 rF1 + rV 50 IM — — — 10.7*Alhydrogel IN = Intranasal administration; OG = Orogastricadministration; IM = Intramuscular administration OMV-V = Bt OMVscontaining Y. pestis V antigen; OMV-F1 = Bt OMVs containing Y. pestis F1antigen; r = Recombinant protein produced in E. coli; *= Average of sixdeterminations; — = not included in the study design. ^(†)The initialdilution of antibody in the assay was 45% hence the limit of the assaywas nominally set at 45%. Sera found to have an ED50 below the limit ofdetection were assigned an ED50 of >45%

There was, however, a high background BCA in many groups of macaques atDay 0. Assessment of the health records of the study subjects did notuncover any unreported health conditions and no veterinary adversehealth observations were made. Although screened and found to be clearof all know high consequence pathogens in primates, a routine rectalswab was able to identify P. aeruginosa in one study participant.Although this organism was not found to be causing disease or inducingany perceivable clinical sign of illness, it is possible that naturalbackground immunity to such a commensal will cross-react with the typethree secretion system (TTSS) components of Y. pestis. The conservationof the TTSS has been discussed and demonstrated in other studies[28-31]. Thus it is postulated that the high background BCA observed insome study animals is due to immunity to type three secretion elementsof commensal bacteria present in the colony. This hypothesis is furthersupported by confirmation by ELISA that there was pre-existing immunityin some primates which cross-reacted with Y. pestis recombinant Vantigen at Day 0 (FIG. 3). This immunity was confirmed in the CE-ELISA(FIG. 7).

Intranasal administration appeared to result in better serumbactericidal responses for both OMV-antigens. All groups immunised withV-OMVs appeared to demonstrate bactericidal activity during the studywith the best response seen in the group immunised with 50 ug OMV-Vintranasally at Day 42 post immunisation. For F1-OMVs the data alsosuggests that intranasal immunisation dose of 25 μg is optimal. WhilstDay 56 BCA data (Table 1) suggests that there was some waning of immunefunctional activity in some groups, at Day 42 almost all groups (exceptthe lowest intranasally dosed F1-OMV group) appeared to demonstratefunctional immunity to Y. pestis.

Discussion

Using bacterial OMVs generated by the bioengineering of the major humancommensal bacterium, Bt, we have successfully developed formulations ofplague vaccine antigens suitable for direct delivery to mucosal sitesincluding the respiratory tract, the site of pneumonic plague infection.Bt OMVs incorporating the V antigen were shown to generate robusthumoral and cell mediated immune responses in both the upper and lowerrespiratory tract, and in the systemic circulation. Using twoindependent surrogate assays of protection, V-OMV elicited immune serapossessed properties important in immune protection, including theability to kill Y. pestis.

Protection against pneumonic plague is the paramount requirement toprevent epidemic spread. An outbreak in Madagascar in 2017 caused inexcess of 2,400 confirmed, probable and suspected cases of plagueincluding more than 200 deaths, with the majority (˜77%) of reportedcases being clinically classified as pneumonic plague [32]. Foremostamongst the virulence factors secreted by Y. pestis are the F1 and Vproteins that are pivotal in preventing phagocytosis and regulating typethree secretion by the bacteria, respectively. When secreted from Y.pestis, V along with other Yersinia outer proteins (Yops) also playsroles in inhibiting cytokine production, platelet aggregation, andapoptosis of macrophages in addition to immune suppression [33]. Whencombined together as purified recombinant proteins V and F1 comprise apowerful candidate vaccine and are amenable to alternative formulationsother than a liquid suspension with alum [34] or alhydrogel [3], whichallows for mucosal or dual route [35] delivery.

Using a prime and single boost oral or nasal immunisation protocol,V-OMVs were effective at inducing antigen specific IgA in mucosal sitesand IgG in the blood with intranasal delivery being the most effectiveroute of administration, particularly for the induction of mucosal IgAresponses. Intranasally delivered V-OMVs were also able to elicit cellmediated immune responses as evidenced by strong recall responses ofPBMCs from immunised animals and the production of pro-inflammatorycytokines. The weaker immunogenicity of V-OMVs delivered via the oralroute may reflect the more hostile environment of the GI-tract and theneed to overcome significant physical (mechanical digestion), chemical(acidic/alkaline pH), biological (enzymes) and microbiological (themicrobiota) barriers prior to accessing inductive immune sites in thelower GI-tract. However, despite these obstacles orally delivered V-OMVswere effective at generating functional antibodies, albeit at lowertitres than that from animals immunised nasally with V-OMVs. At theearliest timepoint of serological analysis at 28 days post immunisation,systemic and mucosal antibody responses were established and increasedover time. In comparison to recombinant protein based vaccines [35] OMVvaccine formulations may therefore be less effective at inducing rapidonset immune responses (within 14 days). If multiple doses are needed toaccelerate the onset and increase the strength and duration of immuneresponses this is less of a problem for OMV vaccines that arenon-invasive and more user-friendly than for injected recombinantprotein vaccines. The effectiveness of N. meningitidis-based OMV vaccineformulations (MenBvac, VA-Mengoc-BC, PorA P1.6-24 and MeNZB) relies on athree or four dose immunisation regimen to provide protection inchildren and adults and control outbreaks of MenB disease [36]. Theoption of increasing the concentration of V antigen in OMV vaccineformulations is not supported by our data that shows that anintermediate dose of antigen (25 μg) intranasally performed as well asand if not better than a two-fold higher dose in terms of generatinghigh tires of both mucosal and systemic antibodies.

The assessment of host immune responses in NHPs was complicated by alevel of pre-existing immunity in some individuals prior to OMVimmunisation. This “background” immunity was confirmed in threeindependent serum antibody assays conducted at three differentlaboratories and sites. Assessment of the health records of the studysubjects in question did not uncover any unreported health conditionsand no veterinary health observations were made. More extensiveinvestigations identified that Pseudomonas aeruginosa was present insome sample swabs (SF unpublished observations). This however, was notaccompanied by symptomatic infection and no veterinary interventionswere required. Antibodies to the type three secretion system and the Vantigen of Y. pestis are known to cross react with that of otherpathogenic Gram negative bacteria including P. aeruginosa as well asVibrio spp. and Aeromonas spp. that encode homologs of the Yersinia Vantigen [28]. Whilst the presence of these bacterial species could notbe confirmed in samples collected during the study, it is possible thatthey infected and were subsequently eliminated by the immune responsethey invoked in these animals at some time prior to this study and thatV-OMV vaccines may have been assisted by such pre-existingcross-reactive immunity. It is noteworthy that this “background”immunity phenomena was not seen in every animal.

An important immune correlate of protection for a candidate vaccine isthe ability to generate neutralising antibodies able to inhibitbacterial killing of host target cells and/or that are cytotoxic and canactively kill the bacteria [3]. Using a novel Y. pestis bactericidalassay, immune sera from F1- and V-OMV immunised NHPs were shown to killbacteria via antibody-dependent cell-mediated cytotoxicity (ADCC) withintranasally administered V-OMVs being particularly effective atgenerating high titres of bactericidal antibodies. Consistent with theability of V-OMVs to generate neutralising antibodies, immune sera fromV-OMV immunised animals contained V-specific antibodies reactive withepitopes of the V antigen bound by a monoclonal anti-V antibody (Mab7.3)that protects mice by passive transfer against plague infection [27].The bactericidal activity of sera from F1-OMV immunised animals isperhaps surprising in view of their weaker immunogenicity and the lowlevels of antigen specific antibodies they generated compared to V-OMVvaccines (FIG. 2). The low levels of F1 expression in Bt, which requiredsensitive LC-MS techniques for detection, may be a consequence ofinherent differences in the translational machinery and requirements forefficient synthesis and/or in secretion sequences used to target newlysynthesised proteins to the periplasm and OMVs in Yersinia versusBacteroides spp. In addition, the inability of Bt to efficientlysynthesise proteins encoded within the caf1 operon, such as caf1M whichencodes a protein that appears to act as a chaperone for F1 with a rolein its post-translational folding and secretion [37], could alsocompromise Bt expression of F1. The inability to detect F1 in F1-OMVlysates using various antibodies in immunoblotting protocols may also beindicative of low levels of expression or the protein not beingexpressed in its native form, or expression of altered structuraldeterminants resulting in the loss of immune epitopes through expressionin Bt and OMVs.

Studies carried out in various animal models including NHPs [32, 16,33-36, 37] indicate that although neutralising antibodies providesprotection against exposure, the development of cell-mediated immunityis essential for protection and clearance of bacteria from the host.Studies using mice with targeted mutations that disrupt Th1 or Th2 CD4 Tcells responses have shown that Th1 driven cell mediated immuneresponses are particularly important in protecting against plague [38].The ability of the V protein to upregulate IL-10 production whichdownregulates the generation of pro-inflammatory cytokines such as TNF□and IFN□ is a key mechanism of virulence and immunosuppression,contributing to the disruption of a balanced Th1/Th2 response thatalongside specific antibodies appears to be optimal for protection [3].In this context the recall response of lymphocytes from V-OMV immunizedanimals that is characterised by the secretion of variouspro-inflammatory cytokines is significant and of predicted benefit inmobilising (MCP-1, IL-8) and activating (IL-1□, IL-6, IL-23) componentsof cell-mediated immune responses in response to plague infection inimmunized animals.

In summary, the key findings from our study are that Bt OMVs can stablyexpress plague antigens, and in particular the V antigen, in thecorrect, immunogenic form and that these engineered OMV vaccineformulations elicit specific immune and antibody responses both in theserum and at mucosal surfaces, including the generation of antibodiesable to kill plague bacteria. Our results also highlight key advantagesour Bt OMV vaccine technology offers over current plague vaccines interms of technology and/or approach. First, OMV vaccine delivery viaoral or nasal administration allows for needle-free, multi-dose deliverythat would enable mass vaccination programs in challenging environmentsand at relatively low cost. Advantageously, this route of immunisationtargets primary sites of mucosal infection as compared to injectablewhole cell or subunit vaccines. Second, compared to subunit or wholecell vaccines the manufacture and re-formulation of OMV vaccines isquicker and can be achieved using readily accessible, relativelyinexpensive technology that has been commercially validated in theproduction of licensed MenB OMV vaccines in current use [39]. Third,patient acceptance is anticipated to be high, requiring out of clinicadministration as compared to injection based vaccines. Fourth, OMVshave intrinsic adjuvanticity and the ability to activate both the innateand adaptive arms of the immune system [21, 22] compared to therequirement for chemical adjuvants such as alum to improveimmunogenicity of subunit vaccines. Fifth, OMV vaccines are acellularand non-infectious increasing their safety compared to live attenuatedor killed whole cell vaccines. Finally, OMVs are stable for ultra-longperiods in liquid and lyophilised form [40] and for several weeks insolution across a wide range of temperatures including 40° C. allowingdistribution to the point of need without cold chain or cold storage,which is particularly advantageous for use in tropical and low incomesettings.

Materials and Methods

Bacteria, Media, Growth Conditions and Transformations

E. coli strains were grown in Luria-Bertani medium at 37° C. Bt strainVPI-5482 and derivative strains were grown under anaerobic conditions at37° C. in BHI medium (Oxoid, UK) supplemented with 0.001% haemin (BHIH)or, with 0.00005% haemin for OMV preparations. Antibiotics were added asselective agents when appropriate: ampicillin 200 μg/ml and erythromycin5 μg/ml. The E. coli strain J53/R751 was supplemented with trimethoprim200 μg/ml when grown for 18 h. E. coli GC10 was transformed byelectroporation using a Gene Pulser II (Bio-Rad, UK). Plasmids weremobilized from E. coli into Bt following a triparental filter matingprotocol [23] using the helper strain J53/R751. Y. pestis strain CO92(biovar Orientalis, NR641, BEI Repositories) was supplied by theBiodefence and Emerging Infections (BEI) Research Repository (USA) inaccordance with International Export and Import Regulatory Requirements.The organism was stored and handled in accordance with US BiologicalSelect Agent or Toxin requirements and was grown using the conditionsdescribed previously [24].

Construction of Yersinia pestis F1- and V1-Antigen Expression Vectors

A synthetic gene construct of 1043 bp encoding the V antigen and asynthetic operon construct of 3826 bp encoding caf1M, caf1A and caf1genes of the caf1 operon that generates the F1 protein were N-terminallyfused to the OmpA signal peptide of Bt to create a construct in silicowith their codon usage being optimised for expression in the samespecies. Signal peptide prediction was obtained by SignalP athttp://www.cbs.dtu.dk/services/SignalP/. During the design of thesynthetic constructs the unique Bacteroides ribosomal binding site [25]required for efficient expression in Bacteroides was accounted for. Theresulting gene cassettes were obtained through gene synthesis andsubsequently cloned into the E. coli plasmid pEX-A2 and pEX-K4 to(Eurofins, Germany), respectively. The cassettes contain BspHI and EcoRIrestriction sites at their 5′ and 3′ ends, respectively, allowing forthe translational fusion of the encoded gene to the start codon in theBacteroides expression vector pGH090 [25]. The genes encoding V1 or F1were excised from the pEX derivatives using BspHI and EcoRI and ligatedinto the NcoI/EcoRI-restricted pGH090 expression vector, resulting inpGH179 and pGH180 respectively. Finally, the sequence integrity of thecloned fragments was verified through sequencing.

Nanoparticle Analysis

Videos were generated using a Nanosight nanoparticle instrument(NanoSight Ltd, Malvern, USA) to count OMV numbers in each OMV sample.Simultaneous measurement of the mean squared displacement of each OMVtracked, the particle diffusion coefficient (D_(t)) and hence sphereequivalent hydrodynamic radius (r_(h)) were determined using theStokes-Einstein equation,

${D_{t} = \frac{k_{B}T}{6\pi\eta r_{h}}},$

where k_(B) is Boltzmann's constant, T is temperature and η is solventviscosity.

Immunoblotting

OMV-V extracts were added to SDS-PAGE loading buffer (NuPage) containingdithiothreitol (Invitrogen). Approximately 7 μg of OMV-V were loadedonto 12% precast Tris-Glycine gels (Novex) and separated byelectrophoresis at 180 volts for 40 min. Gels were transferred onto apolyvinylidene difluoride membrane at 25 volts over 2 h in a solutioncontaining Tris-glycine transfer buffer (Novex). The membrane wasblocked with 10% BSA in TBS (50 mM Tris-HCl, 150 mM NaCl, pH 7.5)-Tween(0.05%) for 30 min at 20° C. Blocking solution was then discarded andthe membrane incubated for 16 h at 4° C. in a 1:1000 dilution of aprimary mouse anti-V antibody (Dstl, UK) in TBS-Tween with 5% BSA. Afterwashing with TBS-Tween 3 times, membranes were incubated 1 h at 20° C.in 5% BSA in TBS-Tween with a 1:1000 dilution of HRP-conjugated goatanti rabbit IgG (ThermoFisher). After 3 washes with TBS-Tween,SuperSignal West Pico chemiluminescent Substrate (ThermoFisher) was usedto detect bound antibody. The detection of F1 in Bt OMV preparations wasdetermined by liquid chromatography and mass spectrometry-(LC-MS) basedproteomics (Proteomics Facility, Univ. Bristol, UK).

Cytokine Analysis

Frozen PBMCs obtained from whole blood by density gradientcentrifugation over Ficoll-Paque Plus (Amersham Biosciences, ChalfontSt. Giles, UK) were thawed, washed twice with RPMI media containing 10%FBS, 2 mM glutamine and 100 U/ml penicillin/streptomycin and adjusted toa concentration of 5×10⁵ cells/ml. Aliquots of 10⁶ cells were platedinto individual U wells of 96 well plates and incubated in triplicatewith media alone or media containing 15 μg/ml rV protein for 72 h at 37°C. in an incubator with 5% CO₂. Control cultures contained media alone.Conditioned media was then harvested and cytokine content determinedusing a bead-based multiplex assay and BD LSRFortessa flow cytometer (BDBiosciences, San Jose, Calif.) according to manufacturer's instructions(BioLegend, San Diego, Calif.).

Antibody ELISA

96 well Nunc-Immuno Microwell plates (Thermo Scientific) were coatedwith 15 μg/ml of Y. pestis V (BG032/VDJPE1) or F (BG032/FD5Pst2)recombinant proteins (Dstl, UK) in ELISA-coating buffer (0.1 M NaHCO3)and incubated for 16 h at 4° C. After washing 3 times with ELISA washbuffer (PBS with 1:2000 Tween-20), plates were blocked for 3 h withblocking buffer (PBS with 2% BSA) at 20° C. with gentle agitation.Serum, saliva, salivary gland and BAL samples were added in dilutionsranging from 1 to 163,840 and the plates were incubated 16 h at 4° C.After 6 washes with ELISA wash buffer, 50 μl of 1:10000 goat anti-monkeyIgG peroxidase conjugated (Sigma: A2054-1 ml) or 1:10000 goatanti-monkey IgA-HRP antibody (Sigma: SAB3700759) were added and plateswere incubated for 1 h at 20° C. with gentle agitation. Plates were thenwashed 6 times with ELISA wash buffer and incubated with 100 μl of TMBHigh Sensitivity Substrate Solution (BioLegend) for 20 min at 20° C., inthe dark. The reaction was stopped by adding 50 μl of 2 N H₂SO₄. Theplates were analysed in a microplate reader at Abs_(450 nm). Absoluteamounts of IgG and IgA antibodies in serum and mucosal samplesrespectively, were determined using a modified ELISA incorporating arange of concentrations of purified monkey IgG (Bio Rad) and IgA (LifeDiagnostics, Inc) and the protocol and reagents described above togenerate standard curves from which IgG and IgA concentrations ofindividual animals were determined.

Bactericidal Assay

This prototype assay was developed in a series of experiments whichoptimised incubation times, heat treatment and complement concentrationusing a standard anti-rF+rV antiserum generated in cynomolgus macaques.Human volunteer and baby rabbit complement (Pellfreeze) were found to besimilar in sensitivity to antibody directed bactericidal activityagainst Y. pestis CO92. Briefly, heat-treated primate sera wereincubated with live Y. pestis bacteria in the presence of 25% v/v rabbitcomplement for X hours with orbital agitation on a 96 well plate shaker.After this incubation, the mixture was plated into blood agar andpermitted to be absorbed into the agar. The plates were then incubatedat 37° C. for two days before manual enumeration was conducted to assessrelative viability. The number of bacteria counted for each test serumantiserum was used to calculate the reduction in counts obtained fromplate spread with the no-antibody, complement control. The dose of testserum (expressed as percentage (v/v) of serum) required to kill 50% oflive Y. pestis CO92 was calculated by interpolation of the dose responsecurved for sample. Each assessment was conducted in duplicate.Bactericidal antibody assessment assays were conducted at PHE on samplestaken on each of the four sample days with serum pools from each of the12 vaccine group. In all studies, the reference standard anti-rV plusanti-rF1 primate serum was also assessed as a means of comparison andmeasure of reproducibility. In all studies, this standard antiserumperformed in a reproducible manner. All controls (including thecomplement only no antibody control) were assessed and found to beperforming as expected.

Competitive ELISA

The detection of antibodies in immune sera from V-OMV immunized animalsable to compete for binding with a monoclonal antibody (Mab7.3) torecombinant V protein was determined in a competitive ELISA (CE-ELISA)assay as previously described [1]. Briefly, rV antigen was coated to96-well microtitre plates (Dynex) at 5 μg/ml in 0.05 ml PBS (16-18 h, 4°C.). After washing, in PBS with 0.02% Tween 20, plates were blocked with0.2 ml 5% w/v skimmed milk powder in PBS (37° C., 1 h). After furtherwashing, 0.05 ml Mab7.3 (1:32,000 in 1% w/v skimmed milk powder in PBS)was added to each well (equivalent to 80 ng/well) and plates wereincubated at 4° C. for 16 h. Normal NHP serum, also diluted to 1:32,000,was added to negative control wells (0.05 ml per well). Plates were thenwashed prior to adding the test serum at a dilution of 1:10 in 1% w/vskimmed milk powder in PBS. A positive control, comprising a referenceserum created by pooling equal aliquots of sera from 4 macaquespreviously parenterally immunized with rF1+ rV antigens and subsequentlysurviving challenge with Y. pestis, was included. Test and control serumsamples were assayed in duplicate. Plates were incubated (1 h, 37° C.)prior to washing and addition of HRP-goat anti-mouse IgG (Serotec;1:2000 in PBS) followed by incubation (37° C., 1 h). Plates were washedprior to addition of ABTS substrate (Sigma) with subsequent reading ofabsorbance at 414 nm. The OD_(414 nm) determined for each test and thereference serum was adjusted by subtraction of the OD_(414 nm)determined for the appropriate control serum. The data were calculatedfrom a titration curve for loss of binding of the mouse antibody, withincreased concentration of human serum.

Animal Experiments

All animal experiments were conducted in full accordance with the AnimalScientific Procedures Act 1986 under UK Home Office approval. Animalexperiments were performed using cynomolgus macaques (Macacafascicularis) that were bred and maintained in animal facilities at PHE,Porton, UK. All animals were free of herpes B-virus, TB, SIV and STLVand were inspected by the Named Veterinary Surgeon prior to entry intothe study. Animals were housed in their existing social groups in penswhich are designed in accordance with the requirements of the UnitedKingdom Home Office Code of Practice for the Housing and Care of AnimalsBred, Supplied or Used for Scientific Purposes, December 2014.

Generation of Standard Reference Antisera.

In order to enable development of a functional biological assay ofrelevance to these studies, antisera to F1 and V were generated byimmunising two cynomolgus macaques with either recombinant F1 (BatchBG032\FD5Pst2, Dstl) or recombinant V (batch BG032\VDJPE1, Dstl) antigenformulated into Alhydrogel adjuvant. Both vaccines were prepared andsupplied to PHE by Dstl. Two primates (one male, one female) wereimmunised with either vaccine on two occasions 3 weeks apart. The doseon each vaccination occasion was 50 μg injected intramuscularly in a 250μl volume. Once an immune response was confirmed to have been inducedvia ELISA, the subjects were humanely euthanised in accordance with a UKHome Office project license and schedule 1 guidance. The sera werecollected after centrifugation of serum separation tubes which hadpermitted the blood to clot. Serum aliquots were created and frozen atbelow −20° C. until required. Stool samples are also collected forreference purposes into OMNIgene GUT OMR-200 tubes.

OMV Vaccines and Vaccination

First, reference anti-V and -F1 antisera were generated by immunisingcynomolgus macaque (n=4 of equal sex) intramuscularly with recombinantpurified F1 and V antigens (50 μg ea.) in a total volume of 250 μl of20% (v/v) alhydrogel. A booster immunisation was given at day 28 withimmune sera obtained at the study end point at day 56. Prior to OMVvaccine immunisations the immune status for each animal with regard toY. pestis antigens were assessed at 14 and 0 days before immunisation.For OMV-V/F1 vaccine studies, groups of animals (n=4/grp of equal sex)were immunised with OMV-V or OMV-F1 vaccine formulations containing12.5, 25 or 50□ g of V/F1 protein via the intranasal route or, with 50μg of V/F1 protein via the oral route in a total volume of 1 ml PBS.Twenty-eight days later all animals received a booster immunisation viathe same route of administration. Blood, stool, saliva and rectal andnasal swab samples were collected at day −14, 0, 28, 42 and at the studyend point of 56 days with body weight, temperature, axillary andinguinal lymph node scores and haemoglobin concentration determined ateach sampling timepoint. At necropsy lung, spleen, liver, heart, lymphnodes, kidney, brain, stomach and intestine were collected forhistopathology. The tissues were examined by light microscopy andevaluated subjectively by a pathologist, blinded to the treatments andgroups in order to prevent bias.

Statistical Analysis

CE-ELISA data were analysed using Graph Pad Prism software v.6 andexpressed as mean±SEM. Statistical comparisons were made using one-wayANOVA or unpaired t-test. The survival data were expressed asKaplan-Meier survival curves and statistical significance was determinedby Log-rank test. P<0.05 was considered statistically significant.

Referring now to the influenza vaccine and Bioengineering human gutcommensal bacteria derived outer membrane vesicles for the delivery toof biologics to the gastrointestinal and respiratory tract moregenerally; we have undertaken a proof-of-principle study to determinethe suitability of using OMVs produced by Bacteroides thetaiotaomicron(Bt), a prominent member of the intestinal microbiota of all animals, todeliver bacteria-, virus- and human-derived proteins to the respiratoryand GI-tract to protect against infection and tissue inflammation andinjury using mouse models of respiratory influenza infection and acuteintestinal colitis. Our findings presented here demonstrate the abilityto express and package within or on the outer membrane of Bt OMVs bothbacteria and virus derived vaccine antigens in a form capable ofeliciting antigen specific immune and antibody responses in mucosaltissues and systemically. Furthermore, immunisation with OMVs containinginfluenza virus vaccine antigens induced heterotypic protection in miceto a 10-fold lethal dose of influenza A virus (IAV). We also show thatOMVs can stably express human therapeutic proteins as exemplified byhuman keratinocyte growth factor-2 (KGF-2) which when delivered orallyreduces disease severity and promotes intestinal epithelial repair andrestitution in animals administered colitis-inducing dextran sodiumsulphate (DSS). Together our data provides evidence of the utility andeffectiveness of using Bt OMVs as a mucosal biologics and drug deliveryplatform technology.

Materials and Methods

Bacteria Strains, Media and Culture

Bacteroides thetaiotaomicron (Bt) and derivate strains (Table 102) weregrown under anaerobic conditions at 37° C. in BHI medium (Oxoid)supplemented with 0.001% haemin (Sigma-Aldrich, St Louis, USA) (BHIH).Antibiotic-resistance markers in B. thetaiotaomicron were selected usingerythromycin (5 μg/ml) and tetracycline (1 μg/ml). E. coli strains weregrown in Luria-Bertani (LB) medium at 37° C. with ampicillin 100 μg/mlor trimethoprim 200 μg/ml for J53 (pR751) strain. Lactococcus lactisUKLc10 and derivative strains were grown in M17 medium (Oxoid)supplemented with 5 g/l glucose at 30° C. Antibiotics were added asselective agents when appropriate: ampicillin 200 μg/ml, erythromycin 5μg/ml and chloramphenicol 10 μg/ml. The E. coli strain J53/R751 wassupplemented with trimethoprim 200 μg/ml when grown for 18 h. E. coliGC10 and L. lactis UKLc10 were transformed by electroporation using aGene Pulser II (Bio-Rad, UK). For constructs relating to pUK200, thehost strain L. lactis UKLc10 was used. Other construction of plasmidsdescribed below were performed using E. coli GC10 as host. Plasmids weremobilized from the E. coli into Bt following a triparental filter matingprotocol [19] using the helper strain J53/R751. Primers used in thisstudy are detailed in Table 102.

Construction of a Bt_3852 Deletion Mutant

A XXX bp chromosomal DNA fragment upstream from Bt_3852 including thefirst 18 nucleotides of its 5′-end region was amplified by PCR using theprimer pair f-5′ompA_SphI, r-5′ompA_SalI. This product was then clonedinto the SpHI/SalI sites of the E. coli-Bacteroides suicide shuttlevector pGH014 [20]. A XXX bp chromosomal DNA fragment downstream fromBT_3852, including the last 46 nucleotides of the 3′-end region, wasamplified by PCR using the primer pair f-3′ompA_BamHI, r-3′ompA_SacI andwas cloned into the BamHI/SacI sites of the pGH014-based plasmid. Theresulting plasmid containing the ΔBT_3852::tetQ construct, was mobilizedfrom E. coli GC10 into Bt by triparental filter mating [19], using E.coli HB101(pRK2013) as the helper strain. Transconjugants were selectedon BHI-haemin agar containing gentamicin (200 mg/L) and tetracycline (1mg/L). Determination of susceptibility to either tetracycline orerythromycin was carried out to identify recombinants that weretetracycline resistant and erythromycin susceptible after re-streakingtransconjugant bacteria on LB-agar containing tetracycline or bothantibiotics. PCR analysis and sequencing were used to confirm theallelic exchange. A transconjugant, GH290, containing the ΔBT_3852::tetQconstruct inserted into the Bt chromosome was selected for furtherstudies.

Generation of Recombinant Bt Strains

Bt Salmonella OmpA/SseB: The Bacteroides expression vector pGH090 [21]was first digested with the restriction enzyme NdeI to remove this siteby Klenow treatment and to create a blunt-ended fragment that was thenreligated. A sequence containing the 90 bp of the Bt_3852 gene 5′ end(encoding a major outer membrane protein, OmpA) corresponding to thesignal peptide sequence (SpOmpA) of the protein obtained from themicrobial genome database (http://mbgd.genome.ad.jp/) was used to designthe complementary oligonucleotide pair SPBTOmpA_fwd and SPBTOmpA_rev.After annealing of the oligonucleotides following a protocol provided byMerck the resulting double-strand DNA contained EcoRI and SpHI 5′overhangs at each end. This linker was cloned into the EcoRI/SpHI sitesof the NdeI deleted version of pGH090, resulting in pGH202 plasmid. The1100 bp ompA and the XXX bp sseB coding region from S. typhimurium wereamplified by PCR from genomic DNA of strain SL1344 using the primerpairs OmpAST_fwd, OmpAST_rev and SseB_fwd, SseB_fwd, respectively. Theresulting fragments were digested with NdeI and EcoRI and cloned intoNdeI/EcoRI-digested pGH202, yielding plasmids pGH182 and pGH183,respectively. The later plasmid was then transformed into E. colicompetent cells (GC10) through electroporation using a Gene Pulser II(Bio-Rad, UK). Successful cloning was checked by sequencing. The plasmidwas mobilized from E. coli to Bt through a triparental mating procedure[19], together with E. coli J53 (pR751) and the correct structure of Btcarrying pGH182 (GH484) was confirmed by sequencing.

Bt IAV: A 635 bp synthetic gene construct encoding a synthetic influenza(H5F; from IAV strain H5N1 (VN/04:A/VietNam/1203/04)) pre-fusionheadless HA mini-stem N-terminally fused to the OmpA signal peptide ofBt was created in silico and its codon usage was optimised forexpression in the same species. The resulting gene cassette was obtainedthrough gene synthesis and subsequently cloned into the E. coli plasmidpEX-K168 (Eurofins, Germany). The cassette contains BspHI and EcoRIrestriction sites at its 5′ and 3′ ends, respectively, allowing for thetranslational fusion of the gene to the start codon in the Bacteroidesexpression vector pGH090 [21]. The gene was excised from pEX-K168 usingBspHI and EcoRI and ligated into the NcoI/EcoRI-restricted pGH090expression vector, resulting in pGH184. Finally the sequence integrityof the cloned fragment was verified through sequencing.

Bt KGF-2: A 581 bp synthetic gene construct encoding the humanfibroblast growth factor-10/keratinocyte growth factor-2 (KGF-2)N-terminally fused to the OmpA signal peptide of Bt was created insilico and its codon usage was optimised for expression in the samespecies. The resulting gene cassette was obtained through gene synthesisand subsequently cloned into the E. coli plasmid pEX-A2 (Eurofins,Germany) as described for the IAV constructs. The final expressionvector was pGH173 with the sequence integrity of the cloned fragmentverified by sequencing.

Expression and Purification of Recombinant StOmpA and StSseB

StOmpA was cloned into His6.tag expression vector pET-15b (Novagen).Briefly, PCR fragments incorporating the coding sequences of ompA andsseB genes were cloned into the NdeI/XhoI restriction sites of pET-15band the resulting plasmids pGH165 and . . . ) transformed into Rosetta2(DE3)pLysS cells yielding strains EcOmpA and EcSseB (Table 102).EcOmpA/SseB cultures were induced at OD_(600 nm) 0.6 by adding 1 mM IPTGfor 5 h after which cells were harvested by centrifugation (5500 g for20 min). The pellet was kept at −20° C. until further use. StOmpA andStSseB proteins were purified under native conditions using protocolsadapted from QIAexpress Ni-NTA Fast Start Handbook (Qiagen) with theamount of protein recovered determined using the Bio-Rad Protein Assay.

OMV Isolation and Characterisation

OMVs were isolated following a method adapted from Stentz et al [20].Briefly, cultures (500 mL) of Bt were centrifuged at 5500 g for 45 minat 4° C. and the supernatants filtered through 0.22 μm pore-sizepolyethersulfone (PES) membranes (Sartorius, Goettingen, Germany) toremove debris and cells. Supernatants were concentrated byultrafiltration (100 kDa molecular weight cut-off, Vivaspin 50R,Sartorius), the retentate was rinsed once with 500 mL of PBS (pH 7.4)and concentrated to 1 mL (approx. 700 μg/ml total protein). The finalOMV suspension were filter sterilized with a 0.22 μm filter. The proteincontent of the final OMV suspensions was determined using the Bio-RadProtein Assay.

The distribution of heterologous proteins within Bt OMVs was establishedin a Proteinase K accessibility/protection assay [20]. Briefly, asuspension of 250 μg of OMVs in 0.1 M phosphate/1 mM EDTA buffer (pH7.0) was incubated for 1 h at 37° C. in the presence of 100 mg/Lproteinase K (Sigma-Aldrich). Proteinase K activity was stopped byaddition of 1 mM phenylmethanesulfonyl fluoride (PMSF) and samplesanalysed by immunoblotting. The Sseb content of Bt OMVs was determinedby targeted proteomics at the University Bristol, UK, ProteomicsFacility.

Nanoparticle Analysis

Videos were generated using a Nanosight nanoparticle instrument(NanoSight Ltd, Malvern, USA) to count OMV numbers in each OMV sample.Simultaneous measurement of the mean squared displacement of each OMVtracked, the particle diffusion coefficient (D_(t)) and hence sphereequivalent hydrodynamic radius (r_(h)) were determined using theStokes-Einstein equation,

${D_{t} = \frac{k_{B}T}{6{\pi\eta}\; r_{h}}},$

where k_(B) is Boltzmann's constant, T is temperature and η is solventviscosity.

Immunoblotting

Bt cell and OMV extracts were obtained by sonication and thesupernatants added to SDS Page loading buffer (NuPage) containingdithiothreitol (Invitrogen). Approximately 7 μg of the total proteinwere loaded onto 12% precast Tris-Glycine gels (Novex) and separated byelectrophoresis at 180 volts for 40 min. The gel was then transferredonto a Polyvinylidene difluoride (PVDF) membrane at 25 volts over 2 h ina solution containing Tris-GLycine Transfer Buffer (Novex). The membranewas blocked with 10% BSA in TBS-Tween (TBS (50 mM Tris-HCl; 150 mM NaCl;pH 7.5) with 0.05% Tween) for 30 min shaking at 20° C. Blocking solutionwas then discarded and the membrane incubated for 16-18 h at 4° C. inTBS-Tween with 5% BSA containing primary antibody (anti-Salmonella OmpA[Antibody Research Corporation], -KGF-2 [ . . . ] or -IAV HA[ . . . ]antibodies). After washing with TBS-Tween, membranes were incubated in5% BSA in TBS-Tween containing HRP-conjugated goat anti rabbit IgG(1:1000 dilution, ThermoFisher) for 1 h at 20° C. After 3 washes withTBS-Tween, SuperSignal West Pico chemiluminescent Substrate(ThermoFisher) was used to detect bound antibody.

Mammalian Cell Culture

The human colonic epithelial cell line Caco-2 (ECACC 86010202) wascultured at 37° C. and 5% CO2 in Dulbecco's Modified Eagle Medium (DMEM)with 4.5 g/L glucose and L-glutamine (Lonza, Switzerland) supplementedwith 5% foetal bovine serum (FBS, Lonza, Switzerland).

Epithelial Cell Scratch Assay

Caco-2 cells were grown in T25 flasks until they reached 90% confluency.Cells were digested using trypsin EDTA (200 mg/L, 170,000 U Trypsin/L,Lonza, Switzerland) and seeded onto 8-well μ-slides (Ibidi, Germany).Cells were grown until they formed a 90% confluent monolayer and thenserum-starved for 8 h. A scratch was performed on the monolayer using asterile tip and cells were washed with PBS to remove cell debris. Theremaining cells were incubated for 72 h in 1% FBS medium supplementedwith heparin (300 μg/mL grade I-A, >180 USP units/ml; Sigma-Aldrich,USA) in the presence of PBS, naïve OMVs, KGF-2 OMVs or recombinant KGF-2(500 ng/mL, PeproTech, USA). Wound healing was monitored by takingimages immediately after scratching, representing a time 0 control, andevery 24 hours using Invertoskop ID03 inverted microscope (Carl Zeiss,Germany) and a Sony Xperia Z5 compact digital camera (Sony, Japan). Themeasurements of the recovered scratch area (pixel²) at each time pointwere analysed using ImageJ software (USA). The experiment was performedin triplicate.

Animal Experiments

Animal experiments were performed using 6 to 8 week old C57BL/6 singlesex mice that were bred and maintained in animal facilities at theUniversity of East Anglia (UK) and University of Liverpool. Mice werehoused in individually ventilated cages and exposed to a 12 h light/darkcycle with free access to a standard laboratory chow diet. Animalexperiments were conducted in full accordance with the Animal ScientificProcedures Act 1986 under UK Home Office approval.

Acute Colitis

The dextran sulphate sodium (DSS) induced mouse model of acute colitiswas used to test the therapeutic potential of KGF-containing OMVs.Groups of male C57BL/6 mice of 8-11 weeks of age were divided into sixgroups (n=5-10/grp) administered either PBS, wild type OMVs, KGF-OMVs,DSS alone, DSS+wild type OMVs or, DSS+KGF-OMVs for 7 days. Experimentalcolitis was induced in the selected groups of mice by administration of2.5% w/v DSS (36,000-50,000 MW, MP Biomedicals, USA) in drinking waterad libitum for 7 days. The other groups of mice received fresh wateralone throughout for the duration of the experiment. PBS and OMVs wereadministered by oral gavage (100 μL) on days 1, 3 and 5 and on day 7mice were euthanized. Fresh faecal pellets were collected daily byplacing individual mice in an empty cage without bedding material for5-15 min. The extent of colitis was evaluated using a disease activityindex (Table S1) made up of determined from daily body weight, stoolconsistency and rectal bleeding assessments. At autopsy the colon wasaseptically extracted and photographed, and the contents collected insterile vials and stored at −80° C. The colon length was measured, andrepresentative samples (0.5 cm length) were taken from the distal regionfor histology by fixing in 10% neutral buffered formalin and embeddingin paraffin. Tissue sections (5 μm) were prepared from each block,stained with 0.5% Mayer's hemalum and Y-eosin solution (H&E, Merck,Germany), and with Alcian blue (Sigma-Aldrich, USA) to visualise gobletcells. Sections were observed under a DMI 3000B microscope at 40×magnification (Leica, Germany) and assessed in a blinded fashion. Thehistological changes were scored (Table S2) and goblet cells enumeratedusing ImageJ software (USA).

OMV Vaccines and Vaccination

For oral immunisation with Salmonella OMV vaccine formulations, 100 μlof StOmpA-OMVs in PBS were administered by oral gavage Boosterimmunisations were given 1 and 2 months later. A control group of micewere immunised via oral gavage with native OMVs. Prior to eachimmunization food was removed for approximately 4 h to decrease stomachacidity. An additional control group of animals were immunised withStOmpA-OMVs via the intraperitoneal route. For intranasal immunisationwith Salmonella and influenza virus OMV vaccine formulations, mice wereanaesthetized then intranasally dosed with either StOmpA OMVs, StSseBOMVs, H5F OMVs, native OMVs or PBS (n=5-10 ea.) and 7 and 14 days laterreceived booster immunizations. For infectious challenge withSalmonella, StOmpA-OMV orally or ip immunised mice were orallyadministered 10⁸ CFU of S. typhimurium SL1344 on day 28 and 5 days laterthe bacterial load in different tissues was determined. For infectiouschallenge with IAV, H5F-OMV immunised mice were on day 28 anaesthetisedand inoculated intra-nasally with 10³ PFU influenza virus strainA/PR/8/34 (PR8, H1N1) in 50 μl sterile PBS. Weights were recoded of eachanimal from the day of challenge up until the end point at 33 days. Atautopsy blood/serum and bronchoalveolar lavage fluid were taken forantibody and cytokine analyses and lung tissue was used to determinevirus titre. For in vivo OMV trafficking studies, mice were intranasallyadministered Dio-labelled H5F-OMVs and 1 and 5 days later OMVacquisition and uptake by macrophage and dendritic cells in the BAL,nasal associated lymphoid tissue (NALT) and cervical and mediastinallymph nodes was determined by flow cytometry.

Virus Quantitation

Plaque assay were performed on homogenates of lung tissue fromPR8-infected mice largely as described previously [22]. Briefly, viralsamples from lungs were titrated in a 10-fold serial dilution from 10¹to 10⁶ in DMEM supplemented with TPCK-trypsin. Each dilution wasincubated with MDCK cells in individual wells of a 24 well plate for 1hour at 37° C., 5% CO₂. The media was aspirated and replaced withoverlay media containing 2.4% Avicel. Plates were incubated at 37° C.,5% CO2 for 72 hours. Avicel was aspirated and plates were washed andcells were fixed in acetone:methanol (60:40) for 10 min. Cells wereallowed to air dry prior to staining with crystal violet for 10 minutes,washed and air dried. Plaques were counted then multiplied by dilutionfactor and volume of virus plated to give viral titre (PFU/ml).

Antibody ELISA

ELISA plates were coated with target antigens (UV inactivated PR8 virus,recombinant Salmonella OmpA or SseB) in 0.1M NaHCO₃ and incubated for12-16 hours at 4° C. Plates were washed 3 times with PBS with 0.05%Tween 20 (PT), incubated with blocking solution (PBS with 2% BSA) for 3h at 20° C., and washed 6 times with PT. BAL and serum samples andsupernatants of homogenised faecal pellets (in phosphate-buffered saline(pH, 7.2) with soybean trypsin inhibitor (0.5 mg/mL; Sigma),phenylmethylsulfonyl fluoride (0.25 mg/mL; Sigma), 0.05 M EDTA, and0.05% Tween 20 (Sigma)) diluted in PBS with 1% BSA, 0.05% Tween (PBT)were added to the plate wells and incubated for 12-16 h at 4° C. thenwashed 6 times with PT and incubated with PBT containing HRP-anti-mouseIgG (1:1000, Thermo-Fisher) or HRP-anti-mouse IgA (1:1000, LifeTechnologies) for 20 min at 20° C. Plates were washed 6 times with PTthen incubated in the dark with TMB High Sensitivity substrate solution(BioLegend) for 30 min at 20° C. The reaction was stopped by theaddition of 2 N H₂SO₄ and the optical density was measured at 450 nmusing a TECAN infinite f50 spectrophotometer (Männedorf, Switzerland).Abcam's IgA Mouse ELISA Kit was used to determine total IgA in SalivaryGlands and Bronchoalveolar lavages (BAL)

Flow Cytometry

Approximately 1×10⁶ tissue-derived cells were incubated in PBS, 2% FCS(PBS-FCS) for 15 min at 4° C. prior to the addition offluorochrome-conjugated monoclonal antibodies specific for CD11b (clone. . . , source), CD11c (clone . . . , source), MHC class II, (clone . .. , source) F4/80 (clone . . . , source), Singlec (clone . . . , source)or CD103 (clone . . . , source) in PBS-FCS and incubation for 30 min at4° C. in the dark. Cells were then washed in PBS-FCS, fixed in PBS, 4%paraformaldehyde for 15 min at 20° C. prior to analysis on a MACSQuantAnalyzer 10 (Miltenyi Biotech UK). Data were analysed using Weaselsoftware (http://www.frankbattye.com.au).

Statistical Analysis

Data were subjected to D'Agostino & Pearson omnibus normality test.One-way ANOVA followed by a Dunnett's multiple comparison post hoc testwas performed using GraphPad Prism 5 software (USA). Statisticallysignificant differences between two mean values were established by ap-value<0.05. Data are presented as the mean±standard deviation (n=10).

Results

Characteristics and Physical Properties of Bt OMVs

Bt is a prominent Gram-negative anaerobe that is universal in nature,occupying multiple and varied habitats including the lower GI-tract ofall vertebrates [23]. In humans it is the most prevalent and abundantbacterial species of the intestinal microbiota [24]. During its growthcycle OMVs bud off from the outer membrane (FIG. 1a ) and are recoveredby a series of filtration and ultracentrifugations of early stationarygrowth phase cultures (see Material and Methods). Bt OMVs range in sizefrom approximately 100 nm to greater than 400 nm with a mean size of 237nm (FIG. 1b ) and retain the characteristic double membrane of theirparental cells (FIG. 1c ). Bt OMVs are highly stable with minimal loss(<10% of total protein or heterologous protein) of luminal proteinsdetected after exposing OMVs to elevated ambient temperature (40° C.),acid, detergent, proteases, sonication and high pressure (FIG. 1d anddata not shown).

Heterologous Bacterial, Viral and Human Proteins Expressed in Bt andIncorporated into OMVs

To test whether the delivery of heterologous antigens into Bt OMVs isbroadly applicable we selected candidate vaccine antigens for theimportant mucosal pathogens (Salmonella typhimurium enterica andinfluenza A virus; IAV) and therapeutic proteins (keratinocyte growthfactor-2; KGF-2). For S. typhi, the outer membrane protein, OmpA, andthe SPI-2 translocon subunit, SseB, were chosen as they elicit antibodyand T cell responses and confer some degree of protective immunity inmice with antibody responses in humans being correlates of immuneprotection [25, 26, 27, 28, 29, 30, 31, 32, 33] For IAV, the H-stalkprotein H5F of an H5N1 (VN/04:A/VietNam/1203/04) strain was selected asit confers robust protection against challenge with multiple IAV strainsand can reduce lung viral titres by 3-fold [34, 35, 36]. KGF-2 isessential for epithelial cell proliferation and preserving the integrityof the intestinal mucosa [36], and has therapeutic potential for thetreatment of inflammatory bowel disease [37].

Mini-genes encoding the bacterial, viral and human proteins were cloneddownstream of sequences encoding N-terminal signal peptides from Btgenes whose products are contained within the lumen or outer membrane ofOMVs (FIG. 2a ) were constructed in L. lactis (for KGF-2) or E. coli(for OmpA, SseB and HF5) hosts and then mobilised into Bt via a triplefilter mating protocol using a help strain. Immunoblotting of whole celland OMV lysates of recombinant Bt strains confirmed expression of allthree heterologous proteins with . . . (FIG. 2c ). The luminal versusouter membrane distribution of the proteins in Bt OMVs was establishedusing a protease protection assay which showed that Salmonella OmpAdistribution was to the outer membrane whereas Salmonella SseB,influenza HF5 and KGF-2 were contained within the lumen of OMVs. WhereasOmpA and H5F were readily detectable in OMV lysates by immunoblotting,SseB was undetectable by this method and instead we relied on proteomicsto confirm its' expression in the lumen of OMVs (data not shown).

Bt OMVs have Inherent Adjuvanticity

Many conventional vaccines rely on the inclusion of adjuvants to enhancetheir immunogenicity and to reduce the number of doses and amount ofantigen (or pathogen component) required to elicit a protective immuneresponse, particularly in immunocompromised people [2, 38]. To formallyevaluate the adjuvant properties of Bt OMVs, mice were administered asingle dose of native OMVs via the intranasal route and 5 days laterlymphoid tissues of the upper and lower respiratory tract (NALT andBALT, respectively) were harvested and analysed by immunohistology forthe presence of organised lymphoid structures and follicles indicativeof an active immune response. The images in FIG. 3 show the presence oflarge organised lymphoid follicles in both the NALT (3 b and d) and BALT(3 c) that contain dendritic cells, T cells and large numbers of Bcells. Of note, OMVs were also effective at eliciting the formation offat associated lymphoid clusters (FALC) in the lung parenchyma (3 e).Consistent with the immune response priming ability of Bt OMVs, within24 h of intranasally administering (fluorescent labelled) OMVs it waspossible to detect them in macrophages and dendritic cells in both thelung and draining cervical and mediastinal lymph nodes (SupplementaryFIG. 1), which are major inductive sites of cell- and humoral-mediatedimmune responses.

From a biosafety perspective, neither orally nor intranasallyadministered native OMVs or vaccine antigen formulated OMVs had noadverse health effects with no tissue pathology evident in treatedanimals at post mortem (data not shown). Orally administered OMVs alsohad no or a small and/or transient effect on intestinal microbes asdetermined from culturing faecal samples on selective media(Supplementary Figure S2).

Based upon these findings and clear evidence of the inherentadjuvanticity of Bt OMVs, we predicted that mucosally administered OMVvaccine formulations will be effective at eliciting both (vaccine)antigen specific antibodies in mucosal sites and systemically.

Mucosal Delivery of OMV Vaccine Formulations

We initially used OMV Salmonella vaccine antigen (St-OmpA and St-SseB)preparations to compare and contrast different formulations and routesof administration. St-OmpA and St-SseB expressing OMVs were administeredto mice via the oral or nasal routes using the dosing regimen depictedin FIG. 4 a. For reference, OMV formulations were also administeredparenterally. At the end of the experiment serum and BAL samples wereanalysed for antigen-specific IgA (mucosal) and IgG (serum) antibodiesby ELISA. For serum antibody responses, the data displayed in FIG. 4bshows that for St-OmpA OMVs the intraperitoneal route of administrationgenerated the highest levels of antigen specific IgG antibodies in theserum compared to the oral or intranasal routes of delivery, for whichcomparable and low levels of St-OmpA specific IgG were detected. ForSt-SseB OMVs, the intranasal route of administration generatedsignificantly higher levels of antigen specific serum IgG antibodiescompared to orally delivered OMVs (p<0.001) that were equivalent to thelevels of antigen-specific IgG seen after intraperitoneal immunisation.For mucosal IgA antibody responses, intranasally administered St-OmpAand St-SseB OMVs were equally effective at eliciting antigen specificIgA antibodies in the lower respiratory tract and BAL (FIG. 4c ). Ofnote, there was more individual variation in the IgA levels and responseto St-OmpA OMVs compared to animals administered St-SseB OMVs. Despitethe induction of Salmonella antigen specific IgG antibodies, neitheroral nor parenteral vaccination with StOmpA-OMVs conferred significantlevels of protection to Salmonella infection as judged by the pathogenburden (CFU) in intestinal and extra-intestinal tissues at the end ofthe study and 5 days post infection (Supplementary Figure S3). Weconcluded therefore that StOmpA-OMV formulated vaccines delivered orally(or parenterally) are unable to protect against oral Salmonellainfection.

Based on the potent adjuvant effect of intranasally administered OMVs(FIG. 3) and higher levels of antigen specific mucosal and systemicantigen specific antibodies after intranasal immunisation with OMV basedvaccines (FIGS. 4b and c ), we investigated further the possibility thatintranasal immunisation with OMV-based vaccines is, compared to oralimmunisation, a better option for conferring protection to infectiouschallenge.

Intranasal OMV Viral Vaccine Formulations Protect Against Pulmonary IAVInfection

HF5 containing OMVs (approximately 0.5 ug/dose) were administeredintranasally to mice followed by two further doses on days 7 and 14.Control mice received either native OMVs or vehicle (PBS) alone. On day28 all groups of mice were given intranasally 10³ PFU of A/PR/8/34 (PR8)H1N1 strain of IAV, which is unrelated to the H5N1 strain of origin ofH5F vaccine antigen. At the end point on day 33, serum and BAL werecollected for assessing anti-viral antibody responses and the lungs wereprocessed for virus titre. During infection the body weight of allgroups of infected animals declined with the greatest weight loss seenin control, PBS administered, animals that exhibited a 20% decrease inweight (FIG. 5a ). Animals immunised with native OMVs displayed a moregradual decline in weight after infection, similar to that in H5F-OMVimmunised animals. Notably, the body weight of the H5F-OMV immunisedanimals increased between day 4 and 5 post-infection indicative of aless severe, and recovery from, infection. This was confirmed from thelung viral titre data (FIG. 5b ). The viral load of animals immunisedwith H5F-OMVs was significantly lower (p<0.006) than that of the othergroups, reflecting an approximate 7 to 8-fold lower level of virus.Consistent with lower viral load in the lungs of H5F-OMV immunisedanimals, BAL H5F specific IgG antibody levels were significantly higher(p=0.004) in this group of animals compared to the other groups (FIG. 5c). Serum anti-H5F IgG antibody levels were also higher in H5F-OMVimmunised animals compared to the other groups although the differencesdid not reach statistical significance (p=0.057).

KGF-2 Containing OMVs Protect Against Acute Colitis

To determine the suitability of Bt OMVs for mucosal delivery oftherapeutic proteins, the human KGF-2 protein was expressed in Bt OMVsby cloning a mini gene containing the coding sequence of the maturehuman kgf-2 gene and the Bt OmpA signal peptide in Bt (Bt-KGF).Immunoblotting of lysates of OMVs harvested from cultures of Bt-KGF(KGF-OMV) established that they contained approximately 5 ug/ml of KGF-2and that the protein was contained within the lumen of OMVs (FIG. 2b ).The biological activity of KGF-OMVs was confirmed in an epithelial cellwounding (scratch) assay in which the addition of intact KGF-OMVs to theepithelial cell cultures promoted epithelial cell proliferation andaccelerated wound closure (Supplementary FIG. 4). KGF-OMVs were testedin the acute murine DSS colitis model, which is a well characterised,simple and reproducible model of intestinal inflammation that isindependent of lymphocyte-mediated responses and in which the clinicalseverity can be quantified and new therapeutic agents evaluated [39].Since DSS primarily affects epithelial cells and inhibits theirproliferation [39] this model is well suited to testing the therapeuticpotential of KGF-OMVs. The design of this study is shown in FIG. 6 a:DSS (2.5% w/v) was added to the drinking water for 7 days after whichmice were given normal drinking water. On days 1, 3 and 5 mice wereorally administered native OMVs or KGF-OMVs (containing approximately0.5 ug KGF/dose), or vehicle (PBS) alone. The dosing regimen was basedin part on our previous studies using a B. ovatus strain engineered toexpress human KGF-2 in vivo that had a therapeutic effect in DSS-colitis[40], and on pilot experiments to assess the tolerability of OMVs (datanot shown).

KGF-OMVs controlled colitis both clinically and pathologically. Weightloss was significantly reduced in animals receiving KGF-OMVs compared tonon-treated animals (p<0.01) or animals administered native OMVs(p<0.001) (FIG. 6b ). KGF-OMVs also reduced the impact of DSS on colonshrinkage and reduction in length (FIGS. 6c and e ), which is anindependent measure of inflammation [41]. Consistent with thetherapeutic effect of KGF-OMVs, disease activity index scores weresignificantly lower in KGF-OMV-treated animals compared to othertreatment groups (FIG. 6d and Supplementary Tables 1 and 2).Histopathology showed that KGF-OMV treatment reduced epithelial damageand inflammatory infiltrate compared to non-treated mice and miceadministered native OMVs (FIG. 7a ). KGF-OMVs also had a beneficialeffect on mucin-producing goblet cells. Compared to non-treated animalsor animals receiving native OMVs there was a significant increase in thenumber of mucin-containing goblet cells in the colonic mucosa of KGF-OMVtreated animals (FIG. 7b ) with the appearance and distribution ofgoblet cells resembling that of control animals receiving water aloneand no DSS (FIG. 7c ).

Discussion

In this study we have provided evidence for the suitability of usingOMVs from the major human gut commensal bacteria, Bt, to deliverbiologics to mucosal sites to protect against infection and injury. Thenanosize and non-replicative status of Bt OMVs together with theirstability and ability to interact with mucosal and systemic host cellsmakes them ideally suited for drug delivery. Moreover, they possessinnate adjuvant properties and the ability to activate immune cells ofboth the innate and adaptive immune system. The use of OMVs fromprominent human commensal bacteria that have established a mutualisticrelationship with, and are well tolerated by, their host is alsodesirable from a safety perspective and in minimising or preventinginappropriate host responses; as evidenced by the absence of any changein health status or pathology in Bt-OMV treated animals.

The Bt OMV technology platform is underpinned by our ability to engineerBacteroides sp. [21, 42, 43] to express heterologous proteins thatretain their biological activity [40, 44, 45], and through the use ofspecific protein secretion sequences to direct them to the periplasmicspace for export and incorporating into the lumen or outer membrane ofBt generated OMVs. Whilst it has not been possible to define the minimumor optimal level of expression of heterologous proteins in Bt OMVsrequired to elicit an appropriate host response, low levels ofexpression only detectable by high resolution LC-MS based proteomics, asfor Salmonella SseB, are sufficient to induce robust host mucosal andsystemic antibody responses. There was therefore no apparent correlationbetween the levels of expression of different proteins within or at thesurface of OMVs and their ability to elicit a host response. Determiningthe amount of biologically active protein in OMV formulations was alsomade difficult by their resilience and ability to resist disruption byhigh pressure, acid, detergent, proteases or sonication. OMV cargo doeshowever become accessible after uptake by host cells [8], most likely asa result of an OMV-intracellular membrane fusion event [12].

The majority of OMV applications have to date focused on vaccinedevelopment [18] as they offer significant advantages over conventionalvaccines; they are non-replicating, provide needle-free delivery, targetmucosal sites, have an established safety record, can elicit innate andantigen-specific adaptive immune responses, possess self-adjuvantproperties (i.e. MAMPs such as LPS), and are relatively cheap andstraightforward to produce. The limitations of current (non-comensal andpathogen-derived) OMV vaccines are the potential for unintended toxicitydue to associated toxins, low expression levels of protective antigens,variable efficacy depending on source and formulation, the need forexogenous adjuvants, and provide only incomplete protection because ofstrain variation. Our work here demonstrates that these limitations canto a large extent be overcome through the use of bioengineered Bt OMVs.The intranasal route of administration was superior to oraladministration in terms of eliciting high levels of Salmonella vaccineantigen-specific mucosal IgA and systemic IgG. This difference mostlikely reflects anatomical differences and the ease and effectiveness ofaccessing host immune inductive sites and their acquisition bymucosa-associated antigen presenting cells in the lower gastrointestinaltract versus the respiratory tract. With smaller distances to travel inthe less harsh environment of the lungs, OMVs can more readily accesshost cells; within 24 h of intranasal administration Bt OMVs wereacquired by macrophages (CD11b⁺, F/480⁺) and dendritic cells (CD11c⁺) inthe mocusa of the upper and lower respiratory tract, with sometrafficking to draining lymph nodes.

The failure to demonstrate protection to infectious challenge in animalsimmunised with Salmonella OMV vaccine formulations could be aconsequence of various factors including sub-optimal expression ofappropriate amounts of immunogenic OmpA antigen in Bt OMVs and thegeneration of sufficient (therapeutic) levels of functional,pathogen-neutralising, antibodies. Although St OmpA was previouslyidentified as a potential cross-species vaccine candidate [46] ourfindings are in line with those of Okamura and colleagues [47] who foundno protection in chickens parenterally immunised with StOmpA. However,the universal adjuvant properties of Bt OMVs suggests they may still beof value in Salmonella vaccine formulations as an adjuvant analogous tomeningococcal OMVs that provide potent adjuvanticity to N. meningitidisprotein vaccines [48, 49].

More compelling evidence for the use of Bt OMV based vaccines wasobtained using OMV-H5F vaccines, which after intranasal administrationconferred a significant level of heterotypic protection against anunrelated strain of IAV. Native OMVs appeared to reduce lung viraltitres compared to non-treated/vaccinated animals which may be relatedto their potent adjuvant properties and the activation of innate andadaptive immune responses, including raised total IgA antibody levels inthe upper and lower respiratory tract that would aid in strengtheningfront line protection against IAV infection [50]. At 5 days postchallenge the average weight of the H5F-OMV immunized animals groupincreased, indicating possible recovery from virus infection, consistentwith a 7-8-fold lower lung viral titre compared to other treatmentgroups. Future refinements to the study protocol should provide aclearer picture of the efficacy of OMV-H5F vaccines in preventing IAVinfection by both homotypic and heterotypic strains of influenza virus.As our study was not an end point study we cannot directly compare thelevel of protection conferred by OMV-H5F vaccines against infectiouschallenge with similar studies trialing OMV-based vaccines; such asthose of Watkins and colleagues [51] that developed E. coli OMV-IAVvaccines and obtained 100% protection in a murine lethal infectiouschallenge model. Collectively, our data provides the rationale andjustification for the continuing development and refinement of the OMVtechnology to improve and optimise their vaccine capabilities andperformance.

Our findings using KGF-containing OMVs to ameliorate experimentalcolitis demonstrates the potential for a broader portfolio ofapplications and in particular, for the mucosal delivery of therapeuticproteins for the treatment of non-infectious, autoimmune-drivenpathologies. Bt OMVs expressing human KGF-2 were effective atameliorating epithelial injury in a chemical (DSS) induced mouse modelof acute colitis, at doses (0.5 ug/dose). The benefit of this form ofdrug delivery is exemplified by comparing the dose required to improvecolonic pathology. The dose of KGF-OMVs (0.5-1.5 ug) used to achieve asignificant reduction in colonic histopathology (FIG. 7a ) is 1-2 ordersof magnitude lower than that required via daily injection (20-100 ug for7 days) to achieve a comparable reduction in colonic pathology [52, 53].Also, the ability to deliver the protein directly to the target tissueusing orally administered OMVs reduces the risk of side effectsassociated with systemic delivery.

In summary, our data adds to the different approaches being developed toexpress heterologous proteins in bacterial microvesicles [18] for avariety of applications and provides evidence for the utility andeffectiveness of using human commensal bacteria as a source ofbioengineered OMVs for the mucosal delivery of different biologics.

TABLE 102 Bacteria strains Protein Antibiotic Species Strain Plasmidexpressed selection* Reference E. coli Rosetta 2(DE3) pLysS pGH165 StOmpA Amp, Cm This study Rosetta 2(DE3) pLysS pGH201 St SseB Amp, Cm Thisstudy Bt VPI-5482 DMSZ Collection GH290 Tet This study GH490 pGH090 Ery[54] GH484 pGH182 St OmpA Ery This study GH486 pGH183 St SseB Ery Thisstudy GH474 pGH173 Hu. KGF-2 Ery This study GH503 pGH184 IAV H5F EryThis study S. typhimurim SL1344 *Amp = ampicillin; Cm = chloramphenicol;Tet = tetracycline; Ery = erythromycin

TABLE 102 Primer sequences Primer Sequence (5′→3′)^(a) f-5′ompA_SphIATCTGCATGCTTTCGAGGAAGAACCGATGGTTGC r-5′ompA_SalIATACGTCGACAATATAGCGGACTGCAATCC f-3′ompA_BamHIACTTGGATCCTTCTGAATCGTGTGGTATTGG r-3′ompA_SacIACTAGAGCTCATCTGTAGAGAAGAAACGGG SPBTOmpA_fwdCATGTTGCTGGCTTTTGCCGGCGTTGCGTCTGTC GCTTCTGCGCAGCAAACCGTGACTGTAACTGAATACGAGGTTATTCATATGTGACG SPBTOmpA_rev AATTCGTCACATATGAATAACCTCGTATTCAGTTACAGTCACGGTTTGCTGCGCAGAAGCGACAGACG CAACGCCGGCAAAAGCCAGCAA OmpAST_fwdTGACCATATGGCTCCGAAAGATAACACC OmpAST_rev GTCAGAATTCTTAAGCCTGCGGCTGAGTTASseB_fwd TGACCATATGTCTTCAGGAAACATCTT SseB_revTGACGAATTCATGAGTACGTTTTCTGCG Xhol_STOmpA_rev ATATCTCGAGGAAACTTAAGCCTGCGGXhol_SseB_rev ATATCTCGAGATGAGTACGTTTTCTGCG

TABLE S1 Disease Activity Index (DAI) criteria and scoring Caecum &Caecum & Weight Stool colon colon contents loss consistency Bleedingappearance appearance Score  <1% Well-formed None Normal Regular shape 0pellets  1-5% White, Irregular but 1 abnormal formed size, strictures 5-10% Loose Slight Random shape 2 10-15% Blood in colon 3 >15%Diarrhoea Gross Blood in 4 caecum

TABLE S2 Colon histology scoring Category Criteria Score Inflammatorycell Severity No infiltration 0 infiltrate Minimal 0-10% 1 Mild 10-25% 2Moderate 26-50% 3 Marked >51% 4 Extent No infiltration 0 Mucosal 1Mucosal and submucosal 2 Mucosal, submucosal and 3 transmural Presenceof oedema Extent No oedema 0 in 0 to 25% of the section 1 in 26 to 50%of the section 2 in more than 51% of the 3 section Epithelial changesGoblet cell None or increase 0 loss Minimal: 0-20% 2 Mild: 21-35% 3Moderate: 36-50% 4 Marked: >50% 5 Erosion Absence 0 Presence 1 Mucosalarchitecture Extent Irregular crypts 4 Crypt loss 5

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1. A vaccine suitable for immunisation against plague or Y. pestisinfection said vaccine comprising outer membrane vesicles (OMVs)including the V and/or F1 antigens of Y. pestis.
 2. A vaccine accordingto claim 1 wherein the OMVs are produced by gram-negative bacteria.
 3. Avaccine according to claim 2 wherein the gram-negative bacteria arehuman commensal gut bacteria.
 4. A vaccine according to claim 3 whereinthe OMVs are produced by bacteria from the genus Bacteroides.
 5. Avaccine according to claim 4 wherein the OMVs are produced byBacteroides thetaiotaomicron (Bt).
 6. A vaccine according to claim 1wherein the genes or mini-genes encoding the V and/or F1 Y. pestisproteins are cloned downstream of sequences encoding the N-terminalsignal peptides of the OMV protein OmpA (BT_3852).
 7. A vaccineaccording to claim 6 wherein the protein products are contained withinthe lumen or outer membrane of OMVs.
 8. A vaccine according to claim 1wherein the gene constructs are generated in E. coli hosts and thenintroduced into Bt.
 9. A vaccine according to claim 1 wherein asynthetic gene construct of 1043 bp encoding the V antigen and/or asynthetic operon construct of 3826 bp encoding caf1M, caf1A and/or caf1genes of the caf1 operon that generates the F1 protein are N-terminallyfused to the OmpA signal peptide of Bt.
 10. A vaccine according to claim9 wherein the resulting gene cassettes were obtained through genesynthesis and subsequently cloned into the E. coli plasmid pEX-A2 andpEX-K4 respectively. 11.-17. (canceled)
 18. A method of producing avaccine or inoculation, said method including the step of introducing atleast part of the gene sequence encoding V and/or F1 antigens of Y.pestis into a gene sequence for OMV production.
 19. (canceled)
 20. Amethod according to claim 18 wherein the vaccine is delivered by oraland/or nasal administration. 21.-22. (canceled)
 23. A vaccine suitablefor immunisation against influenza infection said vaccine comprisingouter membrane vesicles (OMVs) characterised in that within and/or onthe outer membrane of Bt OMVs both bacteria and/or virus derived vaccineantigens are delivered in a form capable of eliciting antigen specificimmune and antibody responses in mucosal tissues and/or systemically.24. A vaccine according to claim 23 wherein Bt OMV is produced using asynthetic gene construct.
 25. A vaccine according to claim 24 whereinthe gene construct encodes a synthetic influenza strain.
 26. A vaccineaccording to claim 25 wherein a 635 bp synthetic gene construct encodinga synthetic influenza (H5F; from IAV strain H5N1(VN/04:A/VietNam/1203/04)) is used.
 27. A vaccine according to claim 26wherein pre-fusion headless HA mini-stem N-terminal is fused to the OmpAsignal peptide of Bt is created in silico.
 28. A vaccine according toclaim 27 wherein the resulting gene cassette is obtained through genesynthesis and subsequently cloned into E. coli.
 29. A vaccine accordingto claim 28 wherein the E. coli plasmid pEX-K168 2 is used.
 30. Avaccine according to claim 29 wherein the cassette contains BspHI andEcoRI restriction sites at its 5′ and 3′ ends, respectively. 31.(canceled)